Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

PubMed

Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

2012-05-01

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

High-Throughput Method for Ranking the Affinity of Peptide Ligands Selected from Phage Display Libraries

PubMed Central

González-Techera, A.; Umpiérrez-Failache, M.; Cardozo, S.; Obal, G.; Pritsch, O.; Last, J. A.; Gee, S. J.; Hammock, B. D.; González-Sapienza, G.

2010-01-01

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major “bottleneck” of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred “en masse” from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide–BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide–BAP protein can find direct application as a tracer reagent. PMID:18393454

Identification of a cardiac specific protein transduction domain by in vivo biopanning using a M13 phage peptide display library in mice.

PubMed

Zahid, Maliha; Phillips, Brett E; Albers, Sean M; Giannoukakis, Nick; Watkins, Simon C; Robbins, Paul D

2010-08-17

A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library. A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain. Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.

Biodiscovery of aluminum binding peptides

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

2013-05-01

Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

PubMed

Nguyen, Kieu T H; Adamkiewicz, Marta A; Hebert, Lauren E; Zygiel, Emily M; Boyle, Holly R; Martone, Christina M; Meléndez-Ríos, Carola B; Noren, Karen A; Noren, Christopher J; Hall, Marilena Fitzsimons

2014-10-01

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display.

PubMed

Hacker, David E; Hoinka, Jan; Iqbal, Emil S; Przytycka, Teresa M; Hartman, Matthew C T

2017-03-17

Highly constrained peptides such as the knotted peptide natural products are promising medicinal agents because of their impressive biostability and potent activity. Yet, libraries of highly constrained peptides are challenging to prepare. Here, we present a method which utilizes two robust, orthogonal chemical steps to create highly constrained bicyclic peptide libraries. This technology was optimized to be compatible with in vitro selections by mRNA display. We performed side-by-side monocyclic and bicyclic selections against a model protein (streptavidin). Both selections resulted in peptides with mid-nanomolar affinity, and the bicyclic selection yielded a peptide with remarkable protease resistance.

Phage display as a technology delivering on the promise of peptide drug discovery.

PubMed

Hamzeh-Mivehroud, Maryam; Alizadeh, Ali Akbar; Morris, Michael B; Church, W Bret; Dastmalchi, Siavoush

2013-12-01

Phage display represents an important approach in the development pipeline for producing peptides and peptidomimetics therapeutics. Using randomly generated DNA sequences and molecular biology techniques, large diverse peptide libraries can be displayed on the phage surface. The phage library can be incubated with a target of interest and the phage which bind can be isolated and sequenced to reveal the displayed peptides' primary structure. In this review, we focus on the 'mechanics' of the phage display process, whilst highlighting many diverse and subtle ways it has been used to further the drug-development process, including the potential for the phage particle itself to be used as a drug carrier targeted to a particular pathogen or cell type in the body. Copyright © 2013 Elsevier Ltd. All rights reserved.

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

Quantitative synthesis of genetically encoded glycopeptide libraries displayed on M13 phage.

PubMed

Ng, Simon; Jafari, Mohammad R; Matochko, Wadim L; Derda, Ratmir

2012-09-21

Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.

Development of a bacteriophage displayed peptide library and biosensor

NASA Astrophysics Data System (ADS)

Chin, Robert C.; Salazar, Noe; Mayo, Michael W.; Villavicencio, Victor I.; Taylor, Richard B.; Chambers, James P.; Valdes, James J.

1996-04-01

A miniaturized, handheld biosensor for identification of hazardous biowarfare agents with high specificity is being developed. An innovative biological recognition system based on bacteriophage displayed peptide receptors will be utilized in conjunction with the miniature biosensor technology being developed. A bacteriophage library has been constructed to provide the artificial receptors. The library can contain millions of bacteriophage with randomly displayed peptide sequences in the phage outer protein coat which act as binding sites for the agents of interest. This library will be used to 'bio-pan' for phages that bind to a number of toxins and infectious agents and can, thus, provide an endless supply of low cost, reliable, specific, and stable artificial receptors. The biosensor instrument will utilize evanescent wave, planar waveguide, far-red dyes, diode laser and miniature circuit technologies for performance and portability.

Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

PubMed

Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

2017-08-01

Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

An integrated vector system for cellular studies of phage display-derived peptides.

PubMed

Voss, Stephan D; DeGrand, Alec M; Romeo, Giulio R; Cantley, Lewis C; Frangioni, John V

2002-09-15

Peptide phage display is a method by which large numbers of diverse peptides can be screened for binding to a target of interest. Even when successful, the rate-limiting step is usually validation of peptide bioactivity using living cells. In this paper, we describe an integrated system of vectors that expedites both the screening and the characterization processes. Library construction and screening is performed using an optimized type 3 phage display vector, mJ(1), which is shown to accept peptide libraries of at least 23 amino acids in length. Peptide coding sequences are shuttled from mJ(1) into one of three families of mammalian expression vectors for cell physiological studies. The vector pAL(1) expresses phage display-derived peptides as Gal4 DNA binding domain fusion proteins for transcriptional activation studies. The vectors pG(1), pG(1)N, and pG(1)C express phage display-derived peptides as green fluorescent protein fusions targeted to the entire cell, nucleus, or cytoplasm, respectively. The vector pAP(1) expresses phage display-derived peptides as fusions to secreted placental alkaline phosphatase. Such enzyme fusions can be used as highly sensitive affinity reagents for high-throughput assays and for cloning of peptide-binding cell surface receptors. Taken together, this system of vectors should facilitate the development of phage display-derived peptides into useful biomolecules.

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

PubMed Central

Yoshida, Kimiko; Goto, Naoko; Ohnami, Shumpei; Aoki, Kazunori

2012-01-01

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine. PMID:23029088

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library.

PubMed

Buzatti, Andréia; Fernandez, Arnielis Diaz; Arenal, Amilcar; Pereira, Erlán; Monteiro, Alda Lucia Gomes; Molento, Marcelo Beltrão

2018-05-24

The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

PubMed

Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

2004-12-01

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peabody, David S.; Chackerian, Bryce; Ashley, Carlee

The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referredmore » to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.« less

Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

PubMed

Thomas, William D; Golomb, Miriam; Smith, George P

2010-12-15

Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. Copyright © 2010 Elsevier Inc. All rights reserved.

Biased selection of propagation-related TUPs from phage display peptide libraries.

PubMed

Zade, Hesam Motaleb; Keshavarz, Reihaneh; Shekarabi, Hosna Sadat Zahed; Bakhshinejad, Babak

2017-08-01

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kay, B. K.; Castagnoli, L.; Biosciences Division

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

99mTc-HYNIC-TNF analogs (WH701) derived from phage display peptide libraries for imaging TNF-receptor-positive ovarian carcinoma: preclinical evaluation

NASA Astrophysics Data System (ADS)

Xiang, Yan; Xia, Jinsong; Wu, H.; Li, H. F.

2002-04-01

Radiolabeled bioactive peptides which bind specifically to surface receptors over expressed in tumor cells are considered as alternatives for tumor detection with ECT. In this investigation, 99mTc-hydrazinonicotinyl - TNF analogs (WH701) was labeled using ethylenediaminediacetic acid (EDDA) as coligand (a number of TNF analogs had been selected and synthesized using random phage-display peptides library in our lab) and Pharmacokinetics and feasibility studies were performed.

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

PhD7Faster: predicting clones propagating faster from the Ph.D.-7 phage display peptide library.

PubMed

Ru, Beibei; 't Hoen, Peter A C; Nie, Fulei; Lin, Hao; Guo, Feng-Biao; Huang, Jian

2014-02-01

Phage display can rapidly discover peptides binding to any given target; thus, it has been widely used in basic and applied research. Each round of panning consists of two basic processes: Selection and amplification. However, recent studies have showed that the amplification step would decrease the diversity of phage display libraries due to different propagation capacity of phage clones. This may induce phages with growth advantage rather than specific affinity to appear in the final experimental results. The peptides displayed by such phages are termed as propagation-related target-unrelated peptides (PrTUPs). They would mislead further analysis and research if not removed. In this paper, we describe PhD7Faster, an ensemble predictor based on support vector machine (SVM) for predicting clones with growth advantage from the Ph.D.-7 phage display peptide library. By using reduced dipeptide composition (ReDPC) as features, an accuracy (Acc) of 79.67% and a Matthews correlation coefficient (MCC) of 0.595 were achieved in 5-fold cross-validation. In addition, the SVM-based model was demonstrated to perform better than several representative machine learning algorithms. We anticipate that PhD7Faster can assist biologists to exclude potential PrTUPs and accelerate the finding of specific binders from the popular Ph.D.-7 library. The web server of PhD7Faster can be freely accessed at http://immunet.cn/sarotup/cgi-bin/PhD7Faster.pl.

Biomathematical Description of Synthetic Peptide Libraries

PubMed Central

Trepel, Martin

2015-01-01

Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org), allowing scientists to plan and analyse their peptide libraries. PMID:26042419

Tumor-targeting peptides from combinatorial libraries*

PubMed Central

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S.

2018-01-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges infighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. PMID:27210583

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Tumor-targeting peptides from combinatorial libraries.

PubMed

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S

2017-02-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges in fighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. Copyright © 2017. Published by Elsevier B.V.

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

PubMed Central

Thomas, William D.; Golomb, Miriam; Smith, George P.

2010-01-01

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

NASA Astrophysics Data System (ADS)

Golec, Piotr; Karczewska-Golec, Joanna; Łoś, Marcin; Węgrzyn, Grzegorz

2012-11-01

Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

Phage display for the discovery of hydroxyapatite-associated peptides.

PubMed

Jin, Hyo-Eon; Chung, Woo-Jae; Lee, Seung-Wuk

2013-01-01

In nature, proteins play a critical role in the biomineralization process. Understanding how different peptide or protein sequences selectively interact with the target crystal is of great importance. Identifying such protein structures is one of the critical steps in verifying the molecular mechanisms of biomineralization. One of the promising ways to obtain such information for a particular crystal surface is to screen combinatorial peptide libraries in a high-throughput manner. Among the many combinatorial library screening procedures, phage display is a powerful method to isolate such proteins and peptides. In this chapter, we will describe our established methods to perform phage display with inorganic crystal surfaces. Specifically, we will use hydroxyapatite as a model system for discovery of apatite-associated proteins in bone or tooth biomineralization studies. This model approach can be generalized to other desired crystal surfaces using the same experimental design principles with a little modification of the procedures. © 2013 Elsevier Inc. All rights reserved.

Biochemical functionalization of peptide nanotubes with phage displayed peptides

NASA Astrophysics Data System (ADS)

Swaminathan, Swathi; Cui, Yue

2016-09-01

The development of a general approach for the biochemical functionalization of peptide nanotubes (PNTs) could open up existing opportunities in both fundamental studies as well as a variety of applications. PNTs are spontaneously assembled organic nanostructures made from peptides. Phage display has emerged as a powerful approach for identifying selective peptide binding motifs. Here, we demonstrate for the first time the biochemical functionalization of PNTs via peptides identified from a phage display peptide library. The phage-displayed peptides are shown to recognize PNTs. These advances further allow for the development of bifunctional peptides for the capture of bacteria and the self-assembly of silver particles onto PNTs. We anticipate that these results could provide significant opportunities for using PNTs in both fundamental studies and practical applications, including sensors and biosensors nanoelectronics, energy storage devices, drug delivery, and tissue engineering.

Identification of antigenic regions on VP2 of African horsesickness virus serotype 3 by using phage-displayed epitope libraries.

PubMed

Bentley, L; Fehrsen, J; Jordaan, F; Huismans, H; du Plessis, D H

2000-04-01

VP2 is an outer capsid protein of African horsesickness virus (AHSV) and is recognized by serotype-discriminatory neutralizing antibodies. With the objective of locating its antigenic regions, a filamentous phage library was constructed that displayed peptides derived from the fragmentation of a cDNA copy of the gene encoding VP2. Peptides ranging in size from approximately 30 to 100 amino acids were fused with pIII, the attachment protein of the display vector, fUSE2. To ensure maximum diversity, the final library consisted of three sub-libraries. The first utilized enzymatically fragmented DNA encoding only the VP2 gene, the second included plasmid sequences, while the third included a PCR step designed to allow different peptide-encoding sequences to recombine before ligation into the vector. The resulting composite library was subjected to immunoaffinity selection with AHSV-specific polyclonal chicken IgY, polyclonal horse immunoglobulins and a monoclonal antibody (MAb) known to neutralize AHSV. Antigenic peptides were located by sequencing the DNA of phages bound by the antibodies. Most antigenic determinants capable of being mapped by this method were located in the N-terminal half of VP2. Important binding areas were mapped with high resolution by identifying the minimum overlapping areas of the selected peptides. The MAb was also used to screen a random 17-mer epitope library. Sequences that may be part of a discontinuous neutralization epitope were identified. The amino acid sequences of the antigenic regions on VP2 of serotype 3 were compared with corresponding regions on three other serotypes, revealing regions with the potential to discriminate AHSV serotypes serologically.

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

PubMed

Tikunova, N V; Morozova, V V

2009-10-01

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

Phage display peptide libraries in molecular allergology: from epitope mapping to mimotope-based immunotherapy.

PubMed

Luzar, J; Štrukelj, B; Lunder, M

2016-11-01

Identification of allergen epitopes is a key component in proper understanding of the pathogenesis of type I allergies, for understanding cross-reactivity and for the development of mimotope immunotherapeutics. Phage particles have garnered recognition in the field of molecular allergology due to their value not only in competitive immunoscreening of peptide libraries but also as immunogenic carriers of allergen mimotopes. They integrate epitope discovery technology and immunization functions into a single platform. This article provides an overview of allergen mimotopes identified through the phage display technique. We discuss the contribution of phage display peptide libraries in determining dominant B-cell epitopes of allergens, in developing mimotope immunotherapy, in understanding cross-reactivity, and in determining IgE epitope profiles of individual patients to improve diagnostics and individualize immunotherapy. We also discuss the advantages and pitfalls of the methodology used to identify and validate the mimotopes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique.

PubMed

You, Fei; Yin, Guangfu; Pu, Ximing; Li, Yucan; Hu, Yang; Huang, Zhongbin; Liao, Xiaoming; Yao, Yadong; Chen, Xianchun

2016-05-01

Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia. Copyright © 2016 Elsevier B.V. All rights reserved.

Specific ligands for classical swine fever virus screened from landscape phage display library.

PubMed

Yin, Long; Luo, Yuzi; Liang, Bo; Wang, Fei; Du, Min; Petrenko, Valery A; Qiu, Hua-Ji; Liu, Aihua

2014-09-01

Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 10(10) transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

PubMed

Cochran, A G; Tong, R T; Starovasnik, M A; Park, E J; McDowell, R S; Theaker, J E; Skelton, N J

2001-01-31

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

Clustering of disulfide-rich peptides provides scaffolds for hit discovery by phage display: application to interleukin-23.

PubMed

Barkan, David T; Cheng, Xiao-Li; Celino, Herodion; Tran, Tran T; Bhandari, Ashok; Craik, Charles S; Sali, Andrej; Smythe, Mark L

2016-11-23

Disulfide-rich peptides (DRPs) are found throughout nature. They are suitable scaffolds for drug development due to their small cores, whose disulfide bonds impart extraordinary chemical and biological stability. A challenge in developing a DRP therapeutic is to engineer binding to a specific target. This challenge can be overcome by (i) sampling the large sequence space of a given scaffold through a phage display library and by (ii) panning multiple libraries encoding structurally distinct scaffolds. Here, we implement a protocol for defining these diverse scaffolds, based on clustering structurally defined DRPs according to their conformational similarity. We developed and applied a hierarchical clustering protocol based on DRP structural similarity, followed by two post-processing steps, to classify 806 unique DRP structures into 81 clusters. The 20 most populated clusters comprised 85% of all DRPs. Representative scaffolds were selected from each of these clusters; the representatives were structurally distinct from one another, but similar to other DRPs in their respective clusters. To demonstrate the utility of the clusters, phage libraries were constructed for three of the representative scaffolds and panned against interleukin-23. One library produced a peptide that bound to this target with an IC 50 of 3.3 μM. Most DRP clusters contained members that were diverse in sequence, host organism, and interacting proteins, indicating that cluster members were functionally diverse despite having similar structure. Only 20 peptide scaffolds accounted for most of the natural DRP structural diversity, providing suitable starting points for seeding phage display experiments. Through selection of the scaffold surface to vary in phage display, libraries can be designed that present sequence diversity in architecturally distinct, biologically relevant combinations of secondary structures. We supported this hypothesis with a proof-of-concept experiment in which three phage libraries were constructed and panned against the IL-23 target, resulting in a single-digit μM hit and suggesting that a collection of libraries based on the full set of 20 scaffolds increases the potential to identify efficiently peptide binders to a protein target in a drug discovery program.

Anti-dengue virus serotype 2 activity and mode of action of a novel peptide.

PubMed

Chew, M-F; Tham, H-W; Rajik, M; Sharifah, S H

2015-10-01

To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action. A phage display peptide library was biopanned against purified DENV-2 and resulted in the identification and selection of a peptide (peptide gg-ww) for further investigation. ELISA was performed, and peptide gg-ww was shown to possess the highest binding affinity against DENV-2. Thus, peptide gg-ww was synthesized for cytotoxicity and antiviral assays. Virus plaque reduction assay, real-time PCR and immunofluorescence assay were used to investigate the inhibitory effect of peptide gg-ww on DENV-2 infection in Vero cells. Three different assays (pre-, simultaneous and post-treatments assays) were performed to investigate the peptide's mode of action. Results indicated that peptide gg-ww possessed strong antiviral activity with a ~96% inhibition rate, which was achieved at 250 μmol l(-1) . Viral replication was inhibited during a simultaneous treatment assay, indicating that the entry of the virus was impeded by this peptide. Peptide gg-ww displayed antiviral action against DENV-2 by targeting an early stage of viral replication (i.e. during viral entry). Peptide gg-ww may represent a new therapeutic candidate for the treatment of DENV infections and is a potential candidate to be developed as a peptide drug. © 2015 The Society for Applied Microbiology.

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

PubMed

Liu, Gary W; Livesay, Brynn R; Kacherovsky, Nataly A; Cieslewicz, Maryelise; Lutz, Emi; Waalkes, Adam; Jensen, Michael C; Salipante, Stephen J; Pun, Suzie H

2015-08-19

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

Screening and identification of RhD antigen mimic epitopes from a phage display random peptide library for the serodiagnosis of haemolytic disease of the foetus and newborn.

PubMed

Wang, Jiao; Song, Jingjing; Zhou, Shuimei; Fu, Yourong; Bailey, Jeffrey A; Shen, Changxin

2018-01-16

Identification of RhD antigen epitopes is a key component in understanding the pathogenesis of haemolytic disease of the foetus and newborn. Research has indicated that phage display libraries are useful tools for identifying novel mimic epitopes (mimotopes) which may help to determine antigen specificity. We selected the mimotopes of blood group RhD antigen by affinity panning a phage display library using monoclonal anti-D. After three rounds of biopanning, positive phage clones were identified by enzyme-linked immunosorbent assay (ELISA) and then sent for sequencing and peptides synthesis. Next, competitive ELISA and erythrocyte haemagglutination inhibition tests were carried out to confirm the inhibitory activity of the synthetic peptide. To evaluate the diagnostic performance of the synthetic peptide, a diagnostic ELISA was examined. Fourteen of 35 phage clones that were chosen randomly from the titering plate were considered to be positive. Following DNA sequencing and translation, 11 phage clones were found to represent the same peptide - RMKMLMMLMRRK (P4) - whereas each of the other three clones represented a unique peptide. Through the competitive ELISA and erythrocyte haemagglutination inhibition tests, the peptide (P4) was verified to have the ability to mimic the RhD antigen. The diagnostic ELISA for P4 proved to be sensitive (82.61%) and specific (88.57%). This study reveals that the P4 peptide can mimic RhD antigen and paves the way for the development of promising targeted diagnostic and therapeutic platforms for haemolytic disease of the foetus and newborn.

Identification and characterization of a salivary-pellicle-binding peptide by phage display.

PubMed

Cukkemane, Nivedita; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Veerman, Enno C I

2014-05-01

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. A library of 2×10(9) phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of bacterial display peptides for use in biosensing applications

NASA Astrophysics Data System (ADS)

Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.

2012-06-01

Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

PubMed

Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

2018-04-25

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potential candidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research. Creative Commons Attribution License

Identification of three novel B-cell epitopes of VMH protein from Vibrio mimicus by screening a phage display peptide library.

PubMed

Xiao, Ning; Cao, Ji; Zhou, Hao; Ding, Shu-Quan; Kong, Ling-Yan; Li, Jin-Nian

2016-12-01

Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish. Copyright © 2016 Elsevier B.V. All rights reserved.

In Vivo 18 FDG/18 Choline Mediated Cerenkov Radiation Energy Transfer (CRET) Multiplexed Optical Imaging for Human Prostate Carcinoma Detection and Staging

DTIC Science & Technology

2016-10-01

human prostate cancer xenografts. We have selected peptides from bacteriophage display libraries that target TF and ErbB2/ErbB3. The peptides have been...facilitate biomarker-specific diagnosis. The specific aims of the proposal are to: 1) select peptides that target the ErbB2/3 heterodimer using novel...parallel in vitro/in vivo phage display techniques; 2) generate NIR-QDs decorated with TF- and ErbB2/3-avid peptides for in vivo molecular

Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination

PubMed Central

Pels, Kevin; Kodadek, Thomas

2015-01-01

The synthesis of libraries of conformationally-constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as peptide tertiary amide (PTA). PTAs are strongly biased conformationally due to allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers. PMID:25695359

Solid-phase synthesis of diverse peptide tertiary amides by reductive amination.

PubMed

Pels, Kevin; Kodadek, Thomas

2015-03-09

The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers.

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

Rousch, Mat; Lutgerink, Jan T; Coote, James; de Bruïne, Adriaan; Arends, Jan-Willem; Hoogenboom, Hennie R

1998-01-01

In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand. PMID:9776337

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

PubMed

Shiuan, David; Chen, Yue-Hao; Lin, Hwan-Kang; Huang, Kao-Jean; Tai, Da-Fu; Chang, Ding-Kwo

2016-06-01

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90 μM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Ligand-regulated peptide aptamers.

PubMed

Miller, Russell A

2009-01-01

The peptide aptamer approach employs high-throughput selection to identify members of a randomized peptide library displayed from a scaffold protein by virtue of their interaction with a target molecule. Extending this approach, we have developed a peptide aptamer scaffold protein that can impart small-molecule control over the aptamer-target interaction. This ligand-regulated peptide (LiRP) scaffold, consisting of the protein domains FKBP12, FRB, and GST, binds to the cell-permeable small-molecule rapamycin and the binding of this molecule can prevent the interaction of the randomizable linker region connecting FKBP12 with FRB. Here we present a detailed protocol for the creation of a peptide aptamer plasmid library, selection of peptide aptamers using the LiRP scaffold in a yeast two-hybrid system, and the screening of those peptide aptamers for a ligand-regulated interaction.

Identification of chondrocyte-binding peptides by phage display.

PubMed

Cheung, Crystal S F; Lui, Julian C; Baron, Jeffrey

2013-07-01

As an initial step toward targeting cartilage tissue for potential therapeutic applications, we sought cartilage-binding peptides using phage display, a powerful technology for selection of peptides that bind to molecules of interest. A library of phage displaying random 12-amino acid peptides was iteratively incubated with cultured chondrocytes to select phage that bind cartilage. The resulting phage clones demonstrated increased affinity to chondrocytes by ELISA, when compared to a wild-type, insertless phage. Furthermore, the selected phage showed little preferential binding to other cell types, including primary skin fibroblast, myocyte and hepatocyte cultures, suggesting a tissue-specific interaction. Immunohistochemical staining revealed that the selected phage bound chondrocytes themselves and the surrounding extracellular matrix. FITC-tagged peptides were synthesized based on the sequence of cartilage-binding phage clones. These peptides, but not a random peptide, bound cultured chondrocytes, and extracelluar matrix. In conclusion, using phage display, we identified peptide sequences that specifically target chondrocytes. We anticipate that such peptides may be coupled to therapeutic molecules to provide targeted treatment for cartilage disorders. Copyright © 2013 Orthopaedic Research Society.

Transmissible gastroenteritis virus; identification of M protein-binding peptide ligands with antiviral and diagnostic potential

USDA-ARS?s Scientific Manuscript database

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and ...

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage-display

PubMed Central

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K.

2012-01-01

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display. PMID:22985966

Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display.

PubMed

Pershad, Kritika; Wypisniak, Karolina; Kay, Brian K

2012-11-23

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious, and time-consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13 the N-terminal Forkhead-associated (FHA1) domain of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be nonfunctional due to misfolding in the bacterial periplasm. To overcome this limitation, we constructed a library of FHA1 variants by mutagenic PCR and isolated functional variants after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1 strand was discovered to be essential for phage display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermally stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20-25mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage display. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

PubMed

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

PubMed

Ohnishi, Toshiyuki; Sakamoto, Kotaro; Asami-Odaka, Asano; Nakamura, Kimie; Shimizu, Ayako; Ito, Takashi; Asami, Taiji; Ohtaki, Tetsuya; Inooka, Hiroshi

2017-01-29

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Advances in synthetic peptides reagent discovery

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Stratis-Cullum, Dimitra N.

2013-05-01

Bacterial display technology offers a number of advantages over competing display technologies (e.g, phage) for the rapid discovery and development of peptides with interaction targeted to materials ranging from biological hazards through inorganic metals. We have previously shown that discovery of synthetic peptide reagents utilizing bacterial display technology is relatively simple and rapid to make laboratory automation possible. This included extensive study of the protective antigen system of Bacillus anthracis, including development of discovery, characterization, and computational biology capabilities for in-silico optimization. Although the benefits towards CBD goals are evident, the impact is far-reaching due to our ability to understand and harness peptide interactions that are ultimately extendable to the hybrid biomaterials of the future. In this paper, we describe advances in peptide discovery including, new target systems (e.g. non-biological materials), advanced library development and clone analysis including integrated reporting.

Mapping protease substrates using a biotinylated phage substrate library.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scholle, M. D.; Kriplani, U.; Pabon, A.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobicmore » is the scissile bond.« less

Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics.

PubMed

Kawakami, Takashi; Ishizawa, Takahiro; Murakami, Hiroshi

2013-08-21

Cyclic structures can increase the proteolytic stability and conformational rigidity of peptides, and N-alkylation of the peptide backbone can make peptides more cell-permeable and resistant to proteolysis. Therefore, cyclic N-alkyl amino acids are expected to be useful building blocks to increase simultaneously these pharmacological properties of peptides. In this study, we screened various cyclic N-alkyl amino acids for their ribosomal incorporation into peptides and identified cyclic N-alkyl amino acids that can be efficiently and successively incorporated. We also demonstrated genetic code reprogramming for reassigning 16 NNU codons to 16 different cyclic N-alkyl amino acids with high fidelity to synthesize highly N-alkylated polycyclic peptidomimetics and an mRNA-displayed library of completely N-alkylated polycyclic peptidomimetics by using our recently developed TRAP (transcription/translation coupled with association of puromycin linker) display. In vitro selection from a highly diverse library of such completely N-alkylated polycyclic peptidomimetics could become a powerful means to discover small-molecule ligands such as drug candidates that can be targeted to biomolecules inside living cells.

Evolution of phage display technology: from discovery to application.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Ahmadzadeh, Vahideh; Akbari, Bahman

2017-03-01

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.

Phage display screening without repetitious selection rounds.

PubMed

't Hoen, Peter A C; Jirka, Silvana M G; Ten Broeke, Bradley R; Schultes, Erik A; Aguilera, Begoña; Pang, Kar Him; Heemskerk, Hans; Aartsma-Rus, Annemieke; van Ommen, Gertjan J; den Dunnen, Johan T

2012-02-15

Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of the immunogenic epitopes of the whole venom component of the Hemiscorpius lepturus scorpion using the phage display peptide library.

PubMed

Jahdasani, Roghaye; Jamnani, Fatemeh Rahimi; Behdani, Mahdi; Habibi-Anbouhi, Mahdi; Yardehnavi, Najmeh; Shahbazzadeh, Delavar; Kazemi-Lomedasht, Fatemeh

2016-12-15

The venom of the Hemiscorpius lepturus scorpion contains mixtures of bioactive compounds that disturb biochemical and physiological functions of the victims. Hemiscorpius lepturus envenomation is recognized as a serious health concern in tropical regions. So far, there is no preventive procedure, and the main focus is on treatment of victims with an antiserum purified from hyper-immunized horses. Although antisera can neutralize the venom, they, in some cases, lead to anaphylactic shock and even death. Selection of peptides mimicking antigenic and immunogenic epitopes of toxins from random peptide libraries is a novel approach for the development of recombinant toxins and poly-epitopic vaccine. To achieve this aim, a phage display peptide library and three rounds of biopanning were performed on immobilized antibodies (IgGs) purified from the sera of hyper-immunized horses. The results show that the highest binding of the phage to immobilized horse antibodies occurred in the third round of biopanning. Over 125 individual clones carrying mimotopes of Hemiscorpius lepturus toxins were selected and subjected for sequencing. The sequencing results identified unique peptides mimicking the antigenic and immunogenic epitopes of Hemiscorpius lepturus toxins. The results of this study provide a basis for further studies and the development of a putative epitopic vaccine and a recombinant toxin. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of peptide sequences that target to the brain using in vivo phage display.

PubMed

Li, Jingwei; Zhang, Qizhi; Pang, Zhiqing; Wang, Yuchen; Liu, Qingfeng; Guo, Liangran; Jiang, Xinguo

2012-06-01

Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200 pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain.

Isolation of a peptide from Ph.D.-C7C phage display library for detection of Cry1Ab.

PubMed

Wang, Yun; Wang, Qian; Wu, Ai-Hua; Hao, Zhen-Ping; Liu, Xian-Jin

2017-12-15

Traditional ELISA methods of using animal immunity yield antibodies for detection Cry toxin. Not only is this incredibly harmful to the animals, but is also time-intensive. Here we developed a simple method to yield the recognition element. Using a critical selection strategy and immunoassay we confirmed a clone from the Ph.D-C7C phage library, which has displayed the most interesting Cry1Ab-binding characteristics examined in this study (Fig. 1). The current study indicates that isolating peptide is an alternative method for the preparation of a recognition element, and that the developed assay is a potentially useful tool for detecting Cry1Ab. Copyright © 2017. Published by Elsevier Inc.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

PubMed

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

PubMed

Solforosi, Laura; Mancini, Nicasio; Canducci, Filippo; Clementi, Nicola; Sautto, Giuseppe Andrea; Diotti, Roberta Antonia; Clementi, Massimo; Burioni, Roberto

2012-07-01

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

PubMed Central

Toledo-Machado, Christina Monerat; Machado de Avila, Ricardo Andrez; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Claudia Martins; Menezes-Souza, Daniel; Carneiro, Rubens Antonio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

2015-01-01

ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL. PMID:25710003

Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom

PubMed Central

Titus, James K; Kay, Matthew K; Glaser, CDR Jacob J

2017-01-01

Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A2 (PLA2) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA2 activity of Western cottonmouth venom in vitro, using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy. PMID:29285351

Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom.

PubMed

Titus, James K; Kay, Matthew K; Glaser, Cdr Jacob J

2017-01-01

Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A 2 (PLA 2 ) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA 2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA 2 activity of Western cottonmouth venom in vitro , using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy.

General M13 phage display: M13 phage display in identification and characterization of protein-protein interactions.

PubMed

Hertveldt, Kirsten; Beliën, Tim; Volckaert, Guido

2009-01-01

In M13 phage display, proteins and peptides are exposed on one of the surface proteins of filamentous phage particles and become accessible to affinity enrichment against a bait of interest. We describe the construction of fragmented whole genome and gene fragment phage display libraries and interaction selection by panning. This strategy allows the identification and characterization of interacting proteins on a genomic scale by screening the fragmented "proteome" against protein baits. Gene fragment libraries allow a more in depth characterization of the protein-protein interaction site by identification of the protein region involved in the interaction.

Engineering RNA phage MS2 virus-like particles for peptide display

NASA Astrophysics Data System (ADS)

Jordan, Sheldon Keith

Phage display is a powerful and versatile technology that enables the selection of novel binding functions from large populations of randomly generated peptide sequences. Random sequences are genetically fused to a viral structural protein to produce complex peptide libraries. From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified by infection of bacteria. For certain applications however, existing phage display platforms have limitations. One such area is in the field of vaccine development, where the goal is to identify relevant epitopes by affinity-selection against an antibody target, and then to utilize them as immunogens to elicit a desired antibody response. Today, affinity selection is usually conducted using display on filamentous phages like M13. This technology provides an efficient means for epitope identification, but, because filamentous phages do not display peptides in the high-density, multivalent arrays the immune system prefers to recognize, they generally make poor immunogens and are typically useless as vaccines. This makes it necessary to confer immunogenicity by conjugating synthetic versions of the peptides to more immunogenic carriers. Unfortunately, when introduced into these new structural environments, the epitopes often fail to elicit relevant antibody responses. Thus, it would be advantageous to combine the epitope selection and immunogen functions into a single platform where the structural constraints present during affinity selection can be preserved during immunization. This dissertation describes efforts to develop a peptide display system based on the virus-like particles (VLPs) of bacteriophage MS2. Phage display technologies rely on (1) the identification of a site in a viral structural protein that is present on the surface of the virus particle and can accept foreign sequence insertions without disruption of protein folding and viral particle assembly, and (2) on the encapsidation of nucleic acid sequences encoding both the VLP and the peptide it displays. The experiments described here are aimed at satisfying the first of these two requirements by engineering efficient peptide display at two different sites in MS2 coat protein. First, we evaluated the suitability of the N-terminus of MS2 coat for peptide insertions. It was observed that random N-terminal 10-mer fusions generally disrupted protein folding and VLP assembly, but by bracketing the foreign sequences with certain specific dipeptides, these defects could be suppressed. Next, the suitability of a coat protein surface loop for foreign sequence insertion was tested. Specifically, random sequence peptides were inserted into the N-terminal-most AB-loop of a coat protein single-chain dimer. Again we found that efficient display required the presence of appropriate dipeptides bracketing the peptide insertion. Finally, it was shown that an N-terminal fusion that tended to interfere specifically with capsid assembly could be efficiently incorporated into mosaic particles when co-expressed with wild-type coat protein.

Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

NASA Astrophysics Data System (ADS)

Essler, Markus; Ruoslahti, Erkki

2002-02-01

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.

Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge

PubMed Central

Kogot, Joshua M.; Zhang, Yanting; Moore, Stephen J.; Pagano, Paul; Stratis-Cullum, Dimitra N.; Chang-Yen, David; Turewicz, Marek; Pellegrino, Paul M.; de Fusco, Andre; Soh, H. Tom; Stagliano, Nancy E.

2011-01-01

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS. PMID:22140433

Biomining of MoS2 with Peptide-based Smart Biomaterials.

PubMed

Cetinel, Sibel; Shen, Wei-Zheng; Aminpour, Maral; Bhomkar, Prasanna; Wang, Feng; Borujeny, Elham Rafie; Sharma, Kumakshi; Nayebi, Niloofar; Montemagno, Carlo

2018-02-20

Biomining of valuable metals using a target specific approach promises increased purification yields and decreased cost. Target specificity can be implemented with proteins/peptides, the biological molecules, responsible from various structural and functional pathways in living organisms by virtue of their specific recognition abilities towards both organic and inorganic materials. Phage display libraries are used to identify peptide biomolecules capable of specifically recognizing and binding organic/inorganic materials of interest with high affinities. Using combinatorial approaches, these molecular recognition elements can be converted into smart hybrid biomaterials and harnessed for biotechnological applications. Herein, we used a commercially available phage-display library to identify peptides with specific binding affinity to molybdenite (MoS 2 ) and used them to decorate magnetic NPs. These peptide-coupled NPs could capture MoS 2 under a variety of environmental conditions. The same batch of NPs could be re-used multiple times to harvest MoS 2 , clearly suggesting that this hybrid material was robust and recyclable. The advantages of this smart hybrid biomaterial with respect to its MoS 2 -binding specificity, robust performance under environmentally challenging conditions and its recyclability suggests its potential application in harvesting MoS 2 from tailing ponds and downstream mining processes.

Phage-display screening identifies LMP1-binding peptides targeting the C-terminus region of the EBV oncoprotein.

PubMed

Ammous-Boukhris, Nihel; Mosbah, Amor; Sahli, Emna; Ayadi, Wajdi; Hadhri-Guiga, Boutheina; Chérif, Ameur; Gargouri, Ali; Mokdad-Gargouri, Raja

2016-11-01

Latent membrane protein 1 (LMP1), a major oncoprotein of Epstein Barr Virus (EBV) is responsible for transforming B lymphocytes in vitro. LMP1 is overexpressed in several EBV-associated malignancies, and different approaches have been developed to reduce its level and accordingly its oncogenic function in tumor tissues. This study aimed to use phage display peptide library to obtain peptides which could specifically bind to the cytoplasmic region of LMP1 to prevent its interaction with signaling proteins. The LMP1 C-terminus region was produced in bacterial E. coli and used as target for the phage library panning. After 3 rounds, 20 phage clones were randomly selected and 8 showed high binding affinity to the recombinant C-terminus LMP1 protein. The most interesting candidates are the FO5 "QPTKDSSPPLRV" and NO4 "STTSPPAVPHNN" peptides since both bind the C-terminus LMP1 as showed by molecular docking. Furthermore, sequence alignment revealed that the FO5 peptide shared sequence similarity with the Death Receptor 4 which belongs to the tumor necrosis factor-related apoptosis-inducing receptor which plays key role in anti-tumor immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell targeting peptides as smart ligands for targeting of therapeutic or diagnostic agents: a systematic review.

PubMed

Mousavizadeh, Ali; Jabbari, Ali; Akrami, Mohammad; Bardania, Hassan

2017-10-01

Cell targeting peptides (CTP) are small peptides which have high affinity and specificity to a cell or tissue targets. They are typically identified by using phage display and chemical synthetic peptide library methods. CTPs have attracted considerable attention as a new class of ligands to delivery specifically therapeutic and diagnostic agents, because of the fact they have several advantages including easy synthesis, smaller physical sizes, lower immunogenicity and cytotoxicity and their simple and better conjugation to nano-carriers and therapeutic or diagnostic agents compared to conventional antibodies. In this systematic review, we will focus on the basic concepts concerning the use of cell-targeting peptides (CTPs), following the approaches of selecting them from peptide libraries. We discuss several developed strategies for cell-specific delivery of different cargos by CTPs, which are designed for drug delivery and diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Selection of peptides binding to metallic borides by screening M13 phage display libraries.

PubMed

Ploss, Martin; Facey, Sandra J; Bruhn, Carina; Zemel, Limor; Hofmann, Kathrin; Stark, Robert W; Albert, Barbara; Hauer, Bernhard

2014-02-10

Metal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials. In this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles. This study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

PubMed

Urban, Johannes H; Moosmeier, Markus A; Aumüller, Tobias; Thein, Marcus; Bosma, Tjibbe; Rink, Rick; Groth, Katharina; Zulley, Moritz; Siegers, Katja; Tissot, Kathrin; Moll, Gert N; Prassler, Josef

2017-11-15

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

A specific RAGE-binding peptide biopanning from phage display random peptide library that ameliorates symptoms in amyloid β peptide-mediated neuronal disorder.

PubMed

Cai, Cuizan; Dai, Xiaoyong; Zhu, Yujie; Lian, Mengyang; Xiao, Fei; Dong, Fangyuan; Zhang, Qihao; Huang, Yadong; Zheng, Qing

2016-01-01

Alzheimer's disease (AD) is an age-related neurodegenerative disorder in which amyloid β (Aβ) peptide accumulates in the brain. The receptor for advanced glycation end product (RAGE) is a cellular binding site for Aβ peptide and mediates amyloid β-induced perturbations in cerebral vessels, neurons, and microglia in AD. Here, we identified a specific high-affinity RAGE inhibitor (APDTKTQ named RP-1) from a phage display library. RP-1 bound to RAGE and inhibited Aβ peptide-induced cellular stress in human neuroblastoma SH-SYSY cells in vitro. Three amino acids in RP-1 are identical to those in the Aβ peptide. RP-1 shows high homology to the 16-23 (KLVFFAED) regions in Aβ peptide and high-affinity RAGE. Functional analyses indicated that RP-1 significantly reduced the level of reactive oxygen species (ROS) and ROS products and that it enhanced catalase and glutathione peroxidase (GPx) activity. Furthermore, it inactivated caspase3 and caspase9 and inhibited the upregulation of RAGE, nuclear factor-κB (NF-κB), and beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) protein expression. In addition, RP-1 activated the PI3K/AKT signaling pathway, inhibiting the interaction between Bax and Bcl-2. Our data suggest that RP-1 is a potent RAGE blocker that effectively controls the progression of Aβ peptide-mediated brain disorders and that it may have potential as a disease-modifying agent for AD.

Discovery and application of peptides that bind to proteins and solid state inorganic materials

NASA Astrophysics Data System (ADS)

Stearns, Linda A.

A series of three projects was undertaken on the theme of peptide-based molecular recognition. In the first project, a messenger RNA (mRNA) display selection was carried out against the II-VI semiconductors zinc sulfide (ZnS), zinc selenide (ZnSe), and cadmium sulfide (CdS). Sequence analysis of 18-mer semiconductor-binding peptides (SBPs) following four rounds of selection indicated that the amino acid sequences were enriched in polar residues compared to the naive library, suggesting that hydrogen-bonding interactions are a dominant mode of interaction between the SBPs and their cognate inorganic surfaces. Select peptides were expressed as fusions of the green fluorescent protein (GFP) to visualize their recognition of semiconductor crystals. Interpretation of the results was complicated by a high fluorescence background that was observed with certain control GFP fusions. Additional experiments, including cross-specificity binding assays, are needed to characterize the peptides that were isolated in this selection. A second project described the practical application of a known inorganic-binding and nucleating peptide. Peptide A3, which was previously isolated by phage display, was chemically conjugated to a short DNA strand using the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC). The resulting peptide-DNA conjugate was hybridized to ten complementary single-stranded capture probes extending outward from the surface of an origami DNA nanotube. A gold precursor solution was added to initiate nucleation and growth of gold nanoparticles at the site of the peptide. Transmission electron microscopy (TEM) was used to visualize the gold nanoparticle-decorated nanostructures. This approach holds immense promise for organizing compositionally-diverse materials at the nanoscale. In a third project, a novel non-iterative approach to mRNA display called covalent capture was demonstrated. Using human transferrin as a target protein, peptides with low-nanomolar affinity were isolated from a combinatorial library of one trillion distinct 12-mer peptide sequences by using UV light to covalently crosslink the peptides to a photoreactive arm that was displayed on the protein surface. The best peptide isolated from this screen exhibited a binding affinity constant (Kd) of 3 nM, which is equivalent to some of the best peptides isolated after many rounds of traditional bead-based selection. The approach itself is general and could be applied to many different types of problems in molecular biology.

Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

PubMed

Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

2014-04-01

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Identification of lanthanum-specific peptides for future recycling of rare earth elements from compact fluorescent lamps.

PubMed

Lederer, Franziska L; Curtis, Susan B; Bachmann, Stefanie; Dunbar, W Scott; MacGillivray, Ross T A

2017-05-01

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO 4 :Ce 3+ ,Tb 3+ (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 10 9 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl 11 O 19 :Tb 3+ ) and BAM (BaMgAl 10 O 17 :Eu 2+ ). No affinity was found for other fluorescent phosphor components such as YOX (Y 2 O 3 :Eu 3+ ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus.

PubMed

Ooi, Diana Jia Miin; Dzulkurnain, Adriya; Othman, Rofina Yasmin; Lim, Saw Hoon; Harikrishna, Jennifer Ann

2006-09-01

A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients.

PubMed

Thong, Kwai-Lin; Tang, Swee-Seong; Tan, Wen-Siang; Devi, Shamala

2007-01-01

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

Discovery of GPX4 inhibitory peptides from random peptide T7 phage display and subsequent structural analysis.

PubMed

Sakamoto, Kotaro; Sogabe, Satoshi; Kamada, Yusuke; Matsumoto, Shin-Ichi; Kadotani, Akito; Sakamoto, Jun-Ichi; Tani, Akiyoshi

2017-01-08

The phospholipid hydroperoxidase glutathione peroxidase (GPX4) is an enzyme that reduces lipid hydroperoxides in lipid membranes. Recently, GPX4 has been investigated as a target molecule that induces iron-dependent cell death (ferroptosis) selectively in cancer cells that express mutant Ras. GPX4 inhibitors have the potential to become novel anti-cancer drugs. However, there are no druggable pockets for conventional small molecules on the molecular surface of GPX4. To generate GPX4 inhibitors, we examined the use of peptides as an alternative to small molecules. By screening peptide libraries displayed on T7 phages, and analyzing the X-ray crystal structures of the peptides, we successfully identified one peptide that binds to near Sec73 of catalytic site and two peptides that bind to another site on GPX4. To our knowledge, this is the first study reporting GPX4 inhibitory peptides and their structural information. Copyright © 2016 Elsevier Inc. All rights reserved.

Protease-Resistant Peptide Ligands from a Knottin Scaffold Library

PubMed Central

Getz, Jennifer A.; Rice, Jeffrey J.; Daugherty, Patrick S.

2011-01-01

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 109 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications. PMID:21615106

Phage display discovery of novel molecular targets in glioblastoma-initiating cells.

PubMed

Liu, J K; Lubelski, D; Schonberg, D L; Wu, Q; Hale, J S; Flavahan, W A; Mulkearns-Hubert, E E; Man, J; Hjelmeland, A B; Yu, J; Lathia, J D; Rich, J N

2014-08-01

Glioblastoma is the most common primary intrinsic brain tumor and remains incurable despite maximal therapy. Glioblastomas display cellular hierarchies with self-renewing glioma-initiating cells (GICs) at the apex. To discover new GIC targets, we used in vivo delivery of phage display technology to screen for molecules selectively binding GICs that may be amenable for targeting. Phage display leverages large, diverse peptide libraries to identify interactions with molecules in their native conformation. We delivered a bacteriophage peptide library intravenously to a glioblastoma xenograft in vivo then derived GICs. Phage peptides bound to GICs were analyzed for their corresponding proteins and ranked based on prognostic value, identifying VAV3, a Rho guanine exchange factor involved tumor invasion, and CD97 (cluster of differentiation marker 97), an adhesion G-protein-coupled-receptor upstream of Rho, as potentially enriched in GICs. We confirmed that both VAV3 and CD97 were preferentially expressed by tumor cells expressing GIC markers. VAV3 expression correlated with increased activity of its downstream mediator, Rac1 (ras-related C3 botulinum toxin substrate 1), in GICs. Furthermore, targeting VAV3 by ribonucleic acid interference decreased GIC growth, migration, invasion and in vivo tumorigenesis. As CD97 is a cell surface protein, CD97 selection enriched for sphere formation, a surrogate of self-renewal. In silico analysis demonstrated VAV3 and CD97 are highly expressed in tumors and inform poor survival and tumor grade, and more common with epidermal growth factor receptor mutations. Finally, a VAV3 peptide sequence identified on phage display specifically internalized into GICs. These results show a novel screening method for identifying oncogenic pathways preferentially activated within the tumor hierarchy, offering a new strategy for developing glioblastoma therapies.

Phage display discovery of novel molecular targets in glioblastoma-initiating cells

PubMed Central

Liu, J K; Lubelski, D; Schonberg, D L; Wu, Q; Hale, J S; Flavahan, W A; Mulkearns-Hubert, E E; Man, J; Hjelmeland, A B; Yu, J; Lathia, J D; Rich, J N

2014-01-01

Glioblastoma is the most common primary intrinsic brain tumor and remains incurable despite maximal therapy. Glioblastomas display cellular hierarchies with self-renewing glioma-initiating cells (GICs) at the apex. To discover new GIC targets, we used in vivo delivery of phage display technology to screen for molecules selectively binding GICs that may be amenable for targeting. Phage display leverages large, diverse peptide libraries to identify interactions with molecules in their native conformation. We delivered a bacteriophage peptide library intravenously to a glioblastoma xenograft in vivo then derived GICs. Phage peptides bound to GICs were analyzed for their corresponding proteins and ranked based on prognostic value, identifying VAV3, a Rho guanine exchange factor involved tumor invasion, and CD97 (cluster of differentiation marker 97), an adhesion G-protein-coupled-receptor upstream of Rho, as potentially enriched in GICs. We confirmed that both VAV3 and CD97 were preferentially expressed by tumor cells expressing GIC markers. VAV3 expression correlated with increased activity of its downstream mediator, Rac1 (ras-related C3 botulinum toxin substrate 1), in GICs. Furthermore, targeting VAV3 by ribonucleic acid interference decreased GIC growth, migration, invasion and in vivo tumorigenesis. As CD97 is a cell surface protein, CD97 selection enriched for sphere formation, a surrogate of self-renewal. In silico analysis demonstrated VAV3 and CD97 are highly expressed in tumors and inform poor survival and tumor grade, and more common with epidermal growth factor receptor mutations. Finally, a VAV3 peptide sequence identified on phage display specifically internalized into GICs. These results show a novel screening method for identifying oncogenic pathways preferentially activated within the tumor hierarchy, offering a new strategy for developing glioblastoma therapies. PMID:24832468

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries.

PubMed

Lamichhane, Tek N; Abeydeera, N Dinuka; Duc, Anne-Cécile E; Cunningham, Philip R; Chow, Christine S

2011-01-28

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m²G966 and m⁵C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low µM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.

Evaluation of Phage Display Discovered Peptides as Ligands for Prostate-Specific Membrane Antigen (PSMA)

PubMed Central

Edwards, W. Barry

2013-01-01

The aim of this study was to identify potential ligands of PSMA suitable for further development as novel PSMA-targeted peptides using phage display technology. The human PSMA protein was immobilized as a target followed by incubation with a 15-mer phage display random peptide library. After one round of prescreening and two rounds of screening, high-stringency screening at the third round of panning was performed to identify the highest affinity binders. Phages which had a specific binding activity to PSMA in human prostate cancer cells were isolated and the DNA corresponding to the 15-mers were sequenced to provide three consensus sequences: GDHSPFT, SHFSVGS and EVPRLSLLAVFL as well as other sequences that did not display consensus. Two of the peptide sequences deduced from DNA sequencing of binding phages, SHSFSVGSGDHSPFT and GRFLTGGTGRLLRIS were labeled with 5-carboxyfluorescein and shown to bind and co-internalize with PSMA on human prostate cancer cells by fluorescence microscopy. The high stringency requirements yielded peptides with affinities KD∼1 µM or greater which are suitable starting points for affinity maturation. While these values were less than anticipated, the high stringency did yield peptide sequences that apparently bound to different surfaces on PSMA. These peptide sequences could be the basis for further development of peptides for prostate cancer tumor imaging and therapy. PMID:23935860

Selection of peptides for binding semiconductor and magnetic materials for the purpose of organizing nanoscaled materials

NASA Astrophysics Data System (ADS)

Whaley, Sandra Renee

A peptide combinatorial approach, also known as phage display, was used to isolate peptides with the ability to bind semiconductor (GaAs, GaN, and InP) and magnetic (Fe2O3 and Fe3O4) materials. The commercially available combinatorial libraries contain randomized peptides either twelve (Ph.D-12(TM)) or seven (Ph.D-C7C(TM)) amino acids in length. The peptides are displayed on the pIII protein of M13 bacteriophage, which have been imaged by atomic force microscopy and transmission electron microscopy. After seven rounds of phage selection with a constrained seven amino acid sequence library (Ph.D-C7C(TM)), two sequences were isolated for binding Fe3O4 (MG-127 and MG-78). The haematite surface was screened with the same library and four unique sequences were isolated after six rounds of selection (HM-95, HM-101, HM-103, and HM-111). According to binding experiments (MG-78 v. MG-127 on Fe3O 4, MG-127 v. HM-95 on Fe3O4 and Fe2O 3, and MG-127 v. HM-95 on gamma-Fe2O3), the MG-127 clone had the highest affinity for iron oxide surfaces (magnetite, haematite, and maghemite) among the clones tested. The Fe3O 4 clone MG-127 displayed the ability to organize Fe3O 4 nanoparticles along bundles of phage. The synthetic peptide analog of this clone was used in the organization of nanoparticles onto the surface of latex beads. The surfaces of the III-V semiconductors were studied using x-ray photoelectron spectroscopy to determine their reactivity in the aqueous conditions used for phage selection. The GaN surface was shown to oxidize the least under these conditions, aiding in the ability to isolate a consensus amino acid sequence responsible for binding to this surface. The G1-3 clone isolated for binding the GaAs (100) surface displayed preferential binding to the GaAs (100) surface over Si (100), GaAs (111) A, GaAs (111) B, and AlGaAs. The synthetic peptide analog of the G12-3 clone was found to preferentially bind to GaAs (100) over either GaAs (111) surfaces or InP (100). This peptide was used to immobilize 10 nm gold particles onto the surface of GaAs within ten minutes. From these results we have shown that it is possible to isolate peptides with high affinities for binding technologically relevant materials, even those not found in nature. These peptides can be used for the organization of pre-formed nanoparticles in solution and on the surface of semiconductor materials.

Bioassembled layered silicate-metal nanoparticle hybrids.

PubMed

Drummy, Lawrence F; Jones, Sharon E; Pandey, Ras B; Farmer, B L; Vaia, Richard A; Naik, Rajesh R

2010-05-01

Here we report on the bioenabled assembly of layered nanohybrids using peptides identified with regard to their affinity to the nanoparticle surface. A dodecamer peptide termed M1, determined from a phage peptide display library, was found to bind to the surface of a layered aluminosilicate (montmorillonite, MMT). Fusion of a metal binding domain to the M1 peptide or the M1 peptide by itself was able to direct the growth of metal nanoparticles, such as gold and cobalt-platinum, respectively, on the MMT. This method of producing hybrid nanoclay materials will have utility in catalytic, optical, biomedical, and composite materials applications.

Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors.

PubMed

Han, Lei; Zhao, Yukun; Cui, Shan; Liang, Bo

2018-06-01

Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.

Biomolecular Recognition of Semiconductors and Magnetic Materials to Assemble Nanoparticle Heterostructures

DTIC Science & Technology

2002-01-01

shown that engineered viruses can recognize specific semiconductor surfaces through the selection by combinatorial phage display method. These specific... phage display libraries. The screening method selected for binding affinity of a population of random peptides displayed as part of the pIII minor coat...shorter spacing than expected distance ( M13 phage length: 880 nm) corresponds to the length scale imposed by the phage which formed the tilted

GuiTope: an application for mapping random-sequence peptides to protein sequences.

PubMed

Halperin, Rebecca F; Stafford, Phillip; Emery, Jack S; Navalkar, Krupa Arun; Johnston, Stephen Albert

2012-01-03

Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda.

PubMed

Pavoni, Emiliano; Vaccaro, Paola; D'Alessio, Valeria; De Santis, Rita; Minenkova, Olga

2013-09-30

Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles.

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2006-07-01

display vectors to allow for both lytic and filamentous versions of the library. For lytic phage display, we chose the T7 -based vector T7Select®10-3b... phage , which do not display a FLAG epitope (data not shown). These data suggest that peptides can be displayed on the surface of T7 using this system...Auto-Antibodies as Breast Cancer Biomarkers To identify auto-antibodies that could be used as breast cancer biomarkers, we are generating a phage

Complementary DNA libraries: an overview.

PubMed

Ying, Shao-Yao

2004-07-01

The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

Biological Nanoplatforms for Self-Assembled Electronics

DTIC Science & Technology

2015-03-24

as M13 , a virus that infects Escherichia coli. Approximately one billion different amino acid sequences are displayed on different viruses in the...sequence when contained within a phage M13 coat protein sequence, not chemically linked to the surface of phage MS2 VLPs. Thus, binding properties may...gallium arsenide in a bacteriophage M13 phage display library, MS2 VLPs modified with the metal binding peptides do not display the same activity

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.

PubMed

Ivarsson, Ylva; Arnold, Roland; McLaughlin, Megan; Nim, Satra; Joshi, Rakesh; Ray, Debashish; Liu, Bernard; Teyra, Joan; Pawson, Tony; Moffat, Jason; Li, Shawn Shun-Cheng; Sidhu, Sachdev S; Kim, Philip M

2014-02-18

The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.

Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier.

PubMed

Uematsu, Shuta; Tabuchi, Yudai; Ito, Yuji; Taki, Masumi

2018-06-01

A peptide-type covalent binder for a target protein was obtained by combinatorial screening of fluoroprobe-conjugated peptide libraries on bacteriophage T7. The solvatochromic fluoroprobe works as a bait during the affinity selection process of phage display. To obtain the targeted covalent binder, the bait in the selected consensus peptide was altered into a reactive warhead possessing a sulfonyl fluoride. The reaction efficiency and site/position specificity of the covalent conjugation between the binder and the target protein were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and rationalized by a protein-ligand docking simulation.

Peptides Derived from a Phage Display Library Inhibit Adhesion and Protect the Host against Infection by Paracoccidioides brasiliensis and Paracoccidioides lutzii

PubMed Central

de Oliveira, Haroldo C.; Michaloski, Jussara S.; da Silva, Julhiany F.; Scorzoni, Liliana; de Paula e Silva, Ana C. A.; Marcos, Caroline M.; Assato, Patrícia A.; Yamazaki, Daniella S.; Fusco-Almeida, Ana M.; Giordano, Ricardo J.; Mendes-Giannini, Maria J. S.

2016-01-01

Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy. PMID:28066254

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nemoto, Naoto, E-mail: nemoto@fms.saitama-u.ac.jp; Innovation Center for Startups, National Institute of Advanced Industrial Science and Technology, 2-2-2 Marunouchi, Chiyoda-ku, Tokyo 100-0005; Janusys Corporation, 508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844

2012-04-27

Highlights: Black-Right-Pointing-Pointer Disulfide-rich peptide aptamer inhibits IL-6-dependent cell proliferation. Black-Right-Pointing-Pointer Disulfide bond of peptide aptamer is essential for its affinity to IL-6R. Black-Right-Pointing-Pointer Inhibitory effect of peptide depends on number and pattern of its disulfide bonds. -- Abstract: Several engineered protein scaffolds have been developed recently to circumvent particular disadvantages of antibodies such as their large size and complex composition, low stability, and high production costs. We previously identified peptide aptamers containing one or two disulfide-bonds as an alternative ligand to the interleukin-6 receptor (IL-6R). Peptide aptamers (32 amino acids in length) were screened from a random peptide library bymore » in vitro peptide selection using the evolutionary molecular engineering method 'cDNA display'. In this report, the antagonistic activity of the peptide aptamers were examined by an in vitro competition enzyme-linked immunosorbent assay (ELISA) and an IL-6-dependent cell proliferation assay. The results revealed that a disulfide-rich peptide aptamer inhibited IL-6-dependent cell proliferation with similar efficacy to an anti-IL-6R monoclonal antibody.« less

Functional Proteomics to Identify Moderators of CD8+ T-Cell Function in Melanoma

DTIC Science & Technology

2013-09-01

could then be used to develop imaging agents that are targeted to the TILs. We used an M13 phage vector, which displays a pentavalent peptide as...of the identified transcript. To directly indentify inhibitory molecules, we have proposed to use phage - display expression libraries to perform...of TCD8. 3. To determine the ligands for molecules that can modulate the TCD8 functional state. Body: A. Phage Screen Summary Phage display

Identification and characterization of epitopes on Plasmodium knowlesi merozoite surface protein-142 (MSP-142) using synthetic peptide library and phage display library.

PubMed

Cheong, Fei Wen; Fong, Mun Yik; Lau, Yee Ling

2016-02-01

Plasmodium knowlesi can cause potentially life threatening human malaria. The Plasmodium merozoite surface protein-142 (MSP-142) is a potential target for malaria blood stage vaccine, and for diagnosis of malaria. Two epitope mapping techniques were used to identify the potential epitopes within P. knowlesi MSP-142. Nine and 14 potential epitopes were identified using overlapping synthetic peptide library and phage display library, respectively. Two regions on P. knowlesi MSP-142 (amino acid residues 37-95 and residues 240-289) were identified to be the potential dominant epitope regions. Two of the prominent epitopes, P10 (TAKDGMEYYNKMGELYKQ) and P31 (RCLLGFKEVGGKCVPASI), were evaluated using mouse model. P10- and P31-immunized mouse sera reacted with recombinant P. knowlesi MSP-142, with the IgG isotype distribution of IgG2b>IgG1>IgG2a>IgG3. Significant higher level of cytokines interferon-gamma and interleukin-2 was detected in P31-immunized mice. Both P10 and P31 could be the suitable epitope candidates to be used in malaria vaccine designs and immunodiagnostic assays, provided further evaluation is needed to validate the potential uses of these epitopes. Copyright © 2015 Elsevier B.V. All rights reserved.

Versatile microbial surface-display for environmental remediation and biofuels production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

2008-02-14

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

The energy landscape of a selective tumor-homing pentapeptide

PubMed Central

Zanuy, David; Flores-Ortega, Alejandra; Casanovas, Jordi; Curco, David; Nussinov, Ruth; Aleman, Carlos

2009-01-01

Recently, a potentially powerful strategy based on the of phage-display libraries has been presented to target tumors via homing peptides attached to nanoparticles. The Cys-Arg-Glu-Lys-Ala (CREKA) peptide sequence has been identified as a tumor-homing peptide that binds to clotted plasmas proteins present in tumor vessels and interstitium. The aim of this work consists of mapping the conformational profile of CREKA to identify the bioactive conformation. For this purpose, a conformational search procedure based on modified Simulated Annealing combined with Molecular Dynamics was applied to three systems that mimic the experimentally used conditions: (i) the free peptide; (ii) the peptide attached to a nanoparticle; and (iii) the peptide inserted in a phage display protein. In addition, the free peptide was simulated in an ionized aqueous solution environment, which mimics the ionic strength of the physiological medium. Accessible minima of all simulated systems reveal a multiple interaction pattern involving the ionized side chains of Arg, Glu and Lys, which induces a β-turn motif in the backbone observed in all simulated CREKA systems. PMID:18588341

Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

PubMed

Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

2012-11-01

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

Ligand-directed profiling of organelles with internalizing phage libraries

PubMed Central

Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

2015-01-01

Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

Identification and Characterization of Strychnine-Binding Peptides Using Phage-Display Screening.

PubMed

Zhang, Fang; Wang, Min; Qiu, Zheng; Wang, Xiao-Meng; Xu, Chun-Lei; Zhang, Xia

2017-01-01

In drug development, phage display is a high-throughput method for identifying the specific cellular targets of drugs. However, insoluble small chemicals remain intractable to this technique because of the difficulty of presenting molecules to phages without occupying or destroying the limited functional groups. In the present study, we selected Strychnine (Stry) as a model compounda and sought to develope an alternative in vitro biopanning strategy against insoluble suspension. A phage library displaying random sequences of fifteen peptides was employed to screen for interactions between Stry and its cellular selective binding peptides, which are of great value to have a complete understanding of the mechanism of Stry for its antitumor activity. After four rounds of biopanning, a selection of 100 binding clones was randomly picked and subjected to modified proliferation and diffusion assays to evaluate the binding affinity of the clones. Finally, eleven clones were identified as positive binders. The corresponding peptides were synthesized and detected for their binding activities using surface plasmon resonance imaging (SPRi). Our study provides a feasible scheme for confirming the interaction of chemical compounds and cellular binding peptides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

[Elaboration of Pseudo-natural Products Using Artificial In Vitro Biosynthesis Systems].

PubMed

Goto, Yuki

2018-01-01

 Peptidic natural products often consist of not only proteinogenic building blocks but also unique non-proteinogenic structures such as macrocyclic scaffolds and N-methylated backbones. Since such non-proteinogenic structures are important structural motifs that contribute to diverse bioactivity, we have proposed that peptides with non-proteinogenic structures should be attractive candidates as artificial bioactive peptides mimicking natural products, or so-called pseudo-natural products. We previously devised an engineered translation system for pseudo-natural peptides, referred to as the flexible in vitro translation (FIT) system. This system enabled "one-pot" synthesis of highly diverse pseudo-natural peptide libraries, which can be rapidly screened by mRNA display technology for the discovery of pseudo-natural peptides with diverse bioactivities.

Direct expression and validation of phage-selected peptide variants in mammalian cells.

PubMed

Quinlan, Brian D; Gardner, Matthew R; Joshi, Vinita R; Chiang, Jessica J; Farzan, Michael

2013-06-28

Phage display is a key technology for the identification and maturation of high affinity peptides, antibodies, and other proteins. However, limitations of bacterial expression restrict the range and sensitivity of assays that can be used to evaluate phage-selected variants. To address this problem, selected genes are typically transferred to mammalian expression vectors, a major rate-limiting step in the iterative improvement of peptides and proteins. Here we describe a system that combines phage display and efficient mammalian expression in a single vector, pDQ1. This system permits immediate expression of phage-selected genes as IgG1-Fc fusions in mammalian cells, facilitating the rapid, sensitive characterization of a large number of library outputs for their biochemical and functional properties. We demonstrate the utility of this system by improving the ability of a CD4-mimetic peptide to bind the HIV-1 envelope glycoprotein and neutralize HIV-1 entry. We further improved the potency of the resulting peptide, CD4mim6, by limiting its ability to induce the CD4-bound conformation of the envelope glycoprotein. Thus, CD4mim6 and its variants can be used to investigate the properties of the HIV-1 envelope glycoprotein, and pDQ1 can accelerate the discovery of new peptides and proteins through phage display.

Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide.

PubMed

Morris, Christopher J; Smith, Mathew W; Griffiths, Peter C; McKeown, Neil B; Gumbleton, Mark

2011-04-10

With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (p<0.05) to the cumulative transport of less than 0.004% for control phage-clone groups. To characterise phage-independent activity of LTP-1 peptide, the LTP-1 peptide was conjugated to a 53kDa anionic PAMAM dendrimer. Compared to respective peptide-dendrimer control conjugates, the LTP-1-PAMAM conjugate displayed a two-fold (bioavailability up to 31%) greater extent of absorption in the IPRL. The LTP-1 peptide-mediated enhancement of transport, when LTP-1 was either attached to the phage clone or conjugated to dendrimer, was sequence-dependent and could be competitively inhibited by co-instillation of excess synthetic free LTP-1 peptide. The specific nature of the target receptor or mechanism involved in LTP-1 lung transport remains unclear although the enhanced transport is enabled through a mechanism that is non-disruptive with respect to the pulmonary transport of hydrophilic permeability probes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ. Copyright © 2010 Elsevier B.V. All rights reserved.

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda

PubMed Central

2013-01-01

Background Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA. Results In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. Conclusions Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles. PMID:24073829

Autotransporter-based cell surface display in Gram-negative bacteria.

PubMed

Nicolay, Toon; Vanderleyden, Jos; Spaepen, Stijn

2015-02-01

Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

PubMed

Warren, Jeremy G; Kasun, George W; Leonard, Takara; Kirkpatrick, Bruce C

2016-05-01

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Targeting the Atypical Chemokine Receptor ACKR3/CXCR7: Phase 1 - Phage Display Peptide Identification and Characterization.

PubMed

Vestal, R D; LaJeunesse, D R; Taylor, E W

2016-01-01

One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's Nterminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, will be the basis for further targeting studies.

Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean

PubMed Central

Wang, Bing; Swaminathan, Sivakumar; Bhattacharyya, Madan K.

2015-01-01

Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed. PMID:26709700

Production of a novel camel single-domain antibody specific for the type III mutant EGFR.

PubMed

Omidfar, K; Rasaee, M J; Modjtahedi, H; Forouzandeh, M; Taghikhani, M; Golmakani, N

2004-01-01

Camelids have a unique immune system capable of producing single-domain heavy-chain antibodies. The antigen-specific domain of these heavy-chain IgGs (VHH) are the smallest binding units produced by the immune system. In this study, we report the isolation and characterization of several binders against the epidermal growth factor receptor (EGFR) vIII retrieved from immune library of camels (Camelus bactrianus and Camelus dromedarius). The EGFRvIII is a ligand-independent, constitutively active, mutated form of the wild-type EGFR. The expression of EGFRvIII has been demonstrated in a wide range of human malignancies, including gliomas, and breast, prostate, ovarian and lung cancer. Camels were immunized with a synthetic peptide corresponding to a mutated sequence and tissue homogenates. Single-domain antibodies (VHH) were directly selected by panning a phage display library on successively decreasing amounts of synthetic peptide immobilized on magnetic beads. The anti-EGFRvIII camel single-domain antibodies selectively bound to the EGFRvIII peptide and reacted specifically with the immunoaffinity-purified antigen from a non-small cell lung cancer patient. These antibodies with affinities in the nanomolar range recognized the EGFRvIII peptide and affinity-purified mutated receptor. We concluded that using the phage display technique, antigen-specific VHH antibody fragments are readily accessible from the camelids. These antibodies may be good candidates for tumor-diagnostic and therapeutic applications. Copyright 2004 S. Karger AG, Basel.

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

PubMed Central

Noppe, Wim; Plieva, Fatima; Galaev, Igor Yu; Pottel, Hans; Deckmyn, Hans; Mattiasson, Bo

2009-01-01

Background Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. Results Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. Conclusion Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection. PMID:19292898

Targeting the prostate for destruction through a vascular address

PubMed Central

Arap, Wadih; Haedicke, Wolfgang; Bernasconi, Michele; Kain, Renate; Rajotte, Daniel; Krajewski, Stanislaw; Ellerby, H. Michael; Bredesen, Dale E.; Pasqualini, Renata; Ruoslahti, Erkki

2002-01-01

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10–15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancer-prone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk. PMID:11830668

Selection, Design and Applications of Solid Binding Peptides for Controlled Biomineralization

NASA Astrophysics Data System (ADS)

Gungormus, Mustafa

The regulated formation of inorganic minerals in living systems is called biomineralization. The mineralization processes of biogenic minerals are strictly regulated by a specific set of biomolecules, typically by proteins. Biologically formed calcium phosphates (hydroxyapatite (HA) in particular) are almost always nano-dimensional and nano-crystalline and are formed in vivo under mild conditions. The importance of understanding the molecular mechanisms underlying biological mineralization is underscored by the diseases associated with ectopic calcification or defects of skeletal mineralization. Proteins that mediate the mineralization process offer us a template for understanding how to potentially enhance the treatment of calcified tissue defects. However, the complexity and the number of proteins involved even in a rather simple biomineralization process can be daunting. The goal of this research was to identify short peptides through peptide-display libraries and demonstrate the utility of these peptides in better understanding the molecular mechanisms underlying biomineralization and as potential therapeutic agents in repairing calcified tissue defects. Using the phage-peptide display library, we have identified peptides that have affinity to HA and can regulate the formation of HA crystals. Utilizing the sequence knowledge obtained, we have identified putative functional domains within amelogenin; a naturally existing mineralization related protein, which, in turn, may provide a better understanding of the formation of dental enamel. These peptides also have the potential to be used to repair calcified tissue defects. To investigate this, we have developed and tested in vitro a hydrogel scaffold with pre-determined functionalities. We have also demonstrated the feasibility of obtaining significant re-mineralization of incipient dental root lesions ex vivo in a relatively short time using the mineralization-directing peptides. The approaches developed through this research may offer clinically acceptable novel procedures to treat calcified tissue defects because the developed peptide is safe to use and promotes the mineralization in a short period of time offered by none of the existing treatment options.

Detection of Listeria monocytogenes with short peptide fragments from class IIa bacteriocins as recognition elements.

PubMed

Azmi, Sarfuddin; Jiang, Keren; Stiles, Michael; Thundat, Thomas; Kaur, Kamaljit

2015-03-09

We employed a direct peptide-bacteria binding assay to screen peptide fragments for high and specific binding to Listeria monocytogenes. Peptides were screened from a peptide array library synthesized on cellulose membrane. Twenty four peptide fragments (each a 14-mer) were derived from three potent anti-listerial peptides, Leucocin A, Pediocin PA1, and Curvacin A, that belong to class IIa bacteriocins. Fragment Leu10 (GEAFSAGVHRLANG), derived from the C-terminal region of Leucocin A, displayed the highest binding among all of the library fragments toward several pathogenic Gram-positive bacteria, including L. monocytogenes, Enterococcus faecalis, and Staphylococcus aureus. The specific binding of Leu10 to L. monocytogenes was further validated using microcantilever (MCL) experiments. Microcantilevers coated with gold were functionalized with peptides by chemical conjugation using a cysteamine linker to yield a peptide density of ∼4.8×10(-3) μmol/cm2 for different peptide fragments. Leu10 (14-mer) functionalized MCL was able to detect Listeria with same sensitivity as that of Leucocin A (37-mer) functionalized MCL, validating the use of short peptide fragments in bacterial detection platforms. Fragment Leu10 folded into a helical conformation in solution, like that of native Leucocin A, suggesting that both Leu10 and Leucocin A may employ a similar mechanism for binding target bacteria. The results show that peptide-conjugated microcantilevers can function as highly sensitive platforms for Listeria detection and hold potential to be developed as biosensors for pathogenic bacteria.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

PubMed

Matsumoto, Yuki; Shindo, Yosuke; Takakusagi, Yoichi; Takakusagi, Kaori; Tsukuda, Senko; Kusayanagi, Tomoe; Sato, Hitoshi; Kawabe, Takumi; Sugawara, Fumio; Sakaguchi, Kengo

2011-12-01

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix. Copyright © 2011 Elsevier Ltd. All rights reserved.

Display technologies: application for the discovery of drug and gene delivery agents

PubMed Central

Sergeeva, Anna; Kolonin, Mikhail G.; Molldrem, Jeffrey J.; Pasqualini, Renata; Arap, Wadih

2007-01-01

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed. PMID:17123658

Identification and characterization of a peptide affinity reagent for detection of noroviruses in clinical samples.

PubMed

Rogers, Jennifer D; Ajami, Nadim J; Fryszczyn, Bartlomiej G; Estes, Mary K; Atmar, Robert L; Palzkill, Timothy

2013-06-01

Norovirus (NoV) is the most common agent of nonbacterial epidemic gastroenteritis and is estimated to cause 21 million cases of the disease in the United States annually. The antigen enzyme-linked immunosorbent assays (ELISAs) currently available for NoV diagnosis detect only certain strains and are approved for use in the United States only in epidemics where NoV is suspected. There is a clear need for simpler, more rapid, and more reliable diagnostic tools for the detection of NoV. In this study, phage display technology was used to screen a library of phage displaying random 12-mer peptides for those that bind to Norwalk virus virus-like particles (NV VLPs). Three phage clones displaying unique peptides were identified, and both the peptide-displaying phages and the peptides were confirmed to bind specifically to NV VLPs. The peptide displayed on phage clone NV-N-R5-1 was determined to bind to the protruding domain of the VP1 capsid protein. This phage also bound to NV VLPs seeded into NoV-negative stool with a limit of detection of 1.56 ng NV VLP. This value was comparable to monoclonal antibody (MAb) 3912, which is currently used in commercially available assays. Furthermore, the NV-N-R5-1 phage exhibited high specificity by detecting NV only in previously characterized NV-positive stool samples in contrast to no detection in NV-negative stool samples. These data demonstrate that the further development of NV-N-R5-1 phage as a diagnostic reagent is possible and might offer several distinct advantages over antibodies, such as decreases in the time and cost of production and ease of isolating phage against other epidemic strains currently circulating as well as those that are emerging.

Discovery of a polystyrene binding peptide isolated from phage display library and its application in peptide immobilization.

PubMed

Qiang, Xu; Sun, Keyong; Xing, Lijun; Xu, Yifeng; Wang, Hong; Zhou, Zhengpin; Zhang, Juan; Zhang, Fang; Caliskan, Bilgen; Wang, Min; Qiu, Zheng

2017-06-01

Phage peptide display is a powerful technique for discovery of various target-specific ligands. However, target-unrelated peptides can often be obtained and cause ambiguous results. Peptide PB-TUP has been isolated repeatedly in our laboratory on different targets and we conducted a research on PB-TUP phage to investigate their binding properties and rate of propagation. ELISA and phage recovery assay demonstrated that PB-TUP phage had a significant superior affinity to polystyrene solid surface compared with control phage clones. In this study, some incidental bindings are excluded like blocking agents and non-specific binding of secondary antibodies. Propagation rate assays of the selected phage clones showed that the growth rate of PB-TUP phage was not superior to the control phages. Furthermore, the binding of PB-TUB to polystyrene was concentration dependent and varied with solution pH. Molecular modeling revealed that stable structures of α-helix and β-turn may contribute to the binding of PB-TUP to polystyrene plate. The PB-TUP sequence was fused to the N-terminus of peptide P2 and the fusion peptide significantly increased the binding affinity to polystyrene. The fusion peptide also enhanced the cell adhesion ability of peptide P2 with human umbilical vein endothelial cell (HUVEC). The addition of the polystyrene binding peptide provided a convenient method for peptide immobilization.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Liu, Yang; Zu, Xiangyang

Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection.more » A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.« less

Recombinant phage probes for Listeria monocytogenes

NASA Astrophysics Data System (ADS)

Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

2007-10-01

Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

Identification of cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage.

PubMed

Sakamoto, Kotaro; Ishibashi, Yoshihiro; Adachi, Ryutaro; Matsumoto, Shin-Ichi; Oki, Hideyuki; Kamada, Yusuke; Sogabe, Satoshi; Zama, Yumi; Sakamoto, Jun-Ichi; Tani, Akiyoshi

2017-08-01

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a K D value of 210nM in SPR analysis and CTPS1-selective inhibition with an IC 50 value of 110nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage

NASA Astrophysics Data System (ADS)

Rothenfluh, Dominique A.; Bermudez, Harry; O'Neil, Conlin P.; Hubbell, Jeffrey A.

2008-03-01

The extracellular matrix of dense, avascular tissues presents a barrier to entry for polymer-based therapeutics, such as drugs encapsulated within polymeric particles. Here, we present an approach by which polymer nanoparticles, sufficiently small to enter the matrix of the targeted tissue, here articular cartilage, are further modified with a biomolecular ligand for matrix binding. This combination of ultrasmall size and biomolecular binding converts the matrix from a barrier into a reservoir, resisting rapid release of the nanoparticles and clearance from the tissue site. Phage display of a peptide library was used to discover appropriate targeting ligands by biopanning on denuded cartilage. The ligand WYRGRL was selected in 94 of 96 clones sequenced after five rounds of biopanning and was demonstrated to bind to collagen II α1. Peptide-functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in the mouse.

The protein and peptide mediated syntheses of non-biologically-produced oxide materials

NASA Astrophysics Data System (ADS)

Dickerson, Matthew B.

Numerous examples exist in nature of organisms which have evolved the ability to produce sophisticated structures composed of inorganic minerals. Studies of such biomineralizing organisms have suggested that specialized biomolecules are, in part, responsible for the controlled formation of these structures. The research detailed in this dissertation is focused on the use of biomolecules (i.e., peptides and proteins) to form non-biologically produced materials under mild reaction conditions (i.e., neutral pH, aqueous solutions, and room temperature). The peptides utilized in the studies detailed in this dissertation were identified through the screening of single crystal rutile TiO2 substrates or Ge powder with a phagedisplayed peptide library. Twenty-one peptides were identified which possessed an affinity for Ge. Three of these twenty one peptides were tested for germania precipitation activity. Those peptides possessing a basic isoelectric point as well as hydroxyl- and imidazole-containing amino acid residues were found to be the most effective in precipitating amorphous germania from an alkoxide precursor. The phage-displayed peptide library screening of TiO2 substrates yielded twenty peptides. Four of these peptides, which were heavily enriched in histidine and/or basic amino acid residues, were found to possess signficant titania precipitation activity. The activity of these peptides was found to correlate with the number of positive charges they carried. The sequence of the most active of the library-identified peptides was modified to yield two additional peptides. The titania precipitation activity of these designed peptides was higher than the parent peptide, with reduced pH dependence. The titania materials generated by the library-identified and designed peptides were found to be composed of amorphous titania as well as <10 nm anatase and/or monoclinic TiO2 crystallites. The production of titania and zirconia resulting from the interaction of the cationic enzyme, hen egg white lysozyme, with Ti- or Zr-lactate precursors is also presented in this dissertation. Lysozyme was found to entrap itself in an active form within the nanoparticles of amorphous titania or zirconia precipitated by this protein under ambient conditions. The lysozyme synthesized titania was observed to be superior to the lysozyme-zirconia materials in preserving the activity of the enzyme under denaturing conditions. Four recombinant proteins, derived from the amino acid sequences of proteins (silaffins) associated with biosilicification in diatoms, were also investigated for titania precipitation activity. The two most basic of these recombinant silaffins, rSil1L and rSilC, were able to induce the formation of titania. The titania precipitates generated by rSil1L were found to be similar to those produced by the phage-displayed library identified peptides. The second recombinant silaffin, rSilC, was found to produce hollow spheres of titania, which, following dehydration, were observed to transform into larger, solid spheres composed of radially aligned columns of rutile TiO2. The highly repetitive nature of the rSilC's amino acid sequence is believed to be responsible for the differences in TiO2 polymorph generated by the different recombinant silaffins and peptides. This dissertation also details research conducted on the formation of titania utilizing rSilC conjugated to synthetic and biogenic silica surfaces. These silica surfaces were functionalized with a newly developed drendritic growth technique. The dendritic functional-group amplification process was demonstrated to increase the loading of hexahisitidine tagged proteins on silica surfaces by more than 40%, as compared to traditional immobilization procedures. The higher loadings of rSilC provided by this dendritic growth method were observed to have a positive impact on the extent of surface mineralization. The titania formed by immobilized rSilC was observed to be composed of amorphous and crystalline TiO2.

The mimic epitopes of Mycobacterium tuberculosis screened by phage display peptide library have serodiagnostic potential for tuberculosis.

PubMed

Wang, Li; Deng, Xiangying; Liu, Haican; Zhao, Lanhua; You, Xiaolong; Dai, Pei; Wan, Kanglin; Zeng, Yanhua

2016-11-01

Mycobacterium tuberculosis is an obligate pathogenic bacterial species in the family of Mycobacteriaceae and attracts excessive immune responses which cause pathology of the lungs in active tuberculosis. The lack of more sensitive and effective diagnosis reagents advocates a further recognition for the fast diagnostic and immunological measures for tuberculosis. Here, two 12-mer peptides with core sequences of SVSVGMKPSPRP (CS1) and TMGFTAPRFPHY (CS2) were screened from a phage display random peptide library using the purified mixed tuberculosis-positive serum as a target. Enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay verified that positive phages exhibited strong binding affinity to mixed tuberculosis-positive serum. BLAST analysis showed that the two sequences may be mimotopes of the Mycobacterium tuberculosis The diagnostic potential for two synthetic mimotope peptides CS1 and CS2 was evaluated using different panels of serum samples (n = 181) by ELISA, and the diagnostic parameters were calculated. CS1 and CS2 achieved sensitivity of 89.41% and 85.88%, and specificities were 90.63% and 87.50%, respectively. We hypothesized that the diagnostic based on CS1 and CS2 may become a promising strategy to enhance the detection of Mycobacterium tuberculosis infection due to higher specificity and sensitivity. Therefore, CS1 and CS2 may possess potentials to provide an experimental basis for the diagnosis of tuberculosis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Organic crystal-binding peptides: morphology control and one-pot formation of protein-displaying organic crystals

NASA Astrophysics Data System (ADS)

Niide, Teppei; Ozawa, Kyohei; Nakazawa, Hikaru; Oliveira, Daniel; Kasai, Hitoshi; Onodera, Mari; Asano, Ryutaro; Kumagai, Izumi; Umetsu, Mitsuo

2015-11-01

Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals.Crystalline assemblies of fluorescent molecules have different functional properties than the constituent monomers, as well as unique optical characteristics that depend on the structure, size, and morphological homogeneity of the crystal particles. In this study, we selected peptides with affinity for the surface of perylene crystal particles by exposing a peptide-displaying phage library in aqueous solution to perylene crystals, eluting the surface-bound phages by means of acidic desorption or liquid-liquid extraction, and amplifying the obtained phages in Escherichia coli. One of the perylene-binding peptides, PeryBPb1: VQHNTKYSVVIR, selected by this biopanning procedure induced perylene molecules to form homogenous planar crystal nanoparticles by means of a poor solvent method, and fusion of the peptide to a fluorescent protein enabled one-pot formation of protein-immobilized crystalline nanoparticles. The nanoparticles were well-dispersed in aqueous solution, and Förster resonance energy transfer from the perylene crystals to the fluorescent protein was observed. Our results show that the crystal-binding peptide could be used for simultaneous control of perylene crystal morphology and dispersion and protein immobilization on the crystals. Electronic supplementary information (ESI) available: Schematic representation of PeryBPb1-fused DsRed-Monomer, fluorescence spectra of perylene crystals and DsRed-Monomer, and emission spectra of DsRed-Monomer at various excitation wavelengths. See DOI: 10.1039/c5nr06471f

Discovery and Development of Synthetic and Natural Biomaterials for Protein Therapeutics and Medical Device Applications

NASA Astrophysics Data System (ADS)

Keefe, Andrew J.

Controlling nonspecific protein interactions is important for applications from medical devices to protein therapeutics. The presented work is a compilation of efforts aimed at using zwitterionic (ionic yet charge neutral) polymers to modify and stabilize the surface of sensitive biomedical and biological materials. Traditionally, when modifying the surface of a material, the stability of the underlying substrate. The materials modified in this dissertation are unique due to their unconventional amorphous characteristics which provide additional challenges. These are poly(dimethyl siloxane) (PDMS) rubber, and proteins. These materials may seem dissimilar, but both have amorphous surfaces, that do not respond well to chemical modification. PDMS is a biomaterial extensively used in medical device manufacturing, but experiences unacceptably high levels of non-specific protein fouling when used with biological samples. To reduce protein fouling, surface modification is often needed. Unfortunately conventional surface modification methods, such as Poly(ethylene glycol) (PEG) coatings, do not work for PDMS due to its amorphous state. Herein, we demonstrate how a superhydrophilic zwitterionic material, poly(carboxybetaine methacrylate) (pCBMA), can provide a highly stable nonfouling coating with long term stability due to the sharp the contrast in hydrophobicity between pCBMA and PDMS. Biological materials, such as proteins, also require stabilization to improve shelf life, circulation time, and bioactivity. Conjugation of proteins with PEG is often used to increase protein stability, but has a detrimental effect on bioactivity. Here we have shown that pCBMA conjugation improves stability in a similar fashion to PEG, but also retains, or even improves, binding affinity due to enhanced protein-substrate hydrophobic interactions. Recognizing that pCBMA chemically resembles the combination of lysine (K) and glutamic acid (E) amino acids, we have shown how zwitterionic nonfouling peptides can be genetically engineered onto a protein to form recombinant protein-polymer conjugates. This technique avoids the need to post-modify proteins, that is often expensive and time consuming in protein manufacturing. Finally, we have developed two new peptide screening methods that were able to select for nonfouling peptide sequences. The selection for nonfouling sequences is not possible using traditional methods (phage display, yeast display, bacterial display and resin display) due to the presence of background interference. In our first nonfouling peptide screening method, we measured the fouling properties of peptides that were immobilized on the surface of solid glass beads. Peptides first needed to be rationally designed, and then subsequently evaluated for protein binding. Using this method, we were able to screen of 10's of sequences. Our second nonfouling peptide screening method is able to screen thousands of peptide sequences using a combinatorially generated peptide library. This was accomplished using controlled pore glass (CPG) beads as substrates to develop one-bead-one-compound (OBOC) peptide libraries. The choice of a porous substrate made it possible to synthesize enough peptide material to allow for peptide sequencing from a single bead using mass spectrometry techniques.

Epitope mapping of PR81 anti-MUC1 monoclonal antibody following PEPSCAN and phage display techniques.

PubMed

Mohammadi, Mohammad; Rasaee, Mohammad Javad; Rajabibazl, Masoumeh; Paknejad, Malihe; Zare, Mehrak; Mohammadzadeh, Sara

2007-08-01

PR81 is an anti-MUC1 monoclonal antibody (MAb) which was generated against human MUC1 mucin that reacted with breast cancerous tissue, MUC1 positive cell line (MCF-7, BT-20, and T-4 7 D), and synthetic peptide, including the tandem repeat sequence of MUC1. Here we characterized the binding properties of PR81 against the tandem repeat of MUC1 by two different epitope mapping techniques, namely, PEPSCAN and phage display. Epitope mapping of PR81 MAb by PEPSCAN revealed a minimal consensus binding sequence, PDTRP, which is found on MUC1 peptide as the most important epitope. Using the phage display peptide library, we identified the motif PD(T/S/G)RP as an epitope and the motif AVGLSPDGSRGV as a mimotope recognized by PR81. Results of these two methods showed that the two residues, arginine and aspartic acid, have important roles in antibody binding and threonine can be substituted by either glycine or serine. These results may be of importance in tailor making antigens used in immunoassay.

Identification of cancer-specific motifs in mimotope profiles of serum antibody repertoire.

PubMed

Gerasimov, Ekaterina; Zelikovsky, Alex; Măndoiu, Ion; Ionov, Yurij

2017-06-07

For fighting cancer, earlier detection is crucial. Circulating auto-antibodies produced by the patient's own immune system after exposure to cancer proteins are promising bio-markers for the early detection of cancer. Since an antibody recognizes not the whole antigen but 4-7 critical amino acids within the antigenic determinant (epitope), the whole proteome can be represented by a random peptide phage display library. This opens the possibility to develop an early cancer detection test based on a set of peptide sequences identified by comparing cancer patients' and healthy donors' global peptide profiles of antibody specificities. Due to the enormously large number of peptide sequences contained in global peptide profiles generated by next generation sequencing, the large number of cancer and control sera is required to identify cancer-specific peptides with high degree of statistical significance. To decrease the number of peptides in profiles generated by nextgen sequencing without losing cancer-specific sequences we used for generation of profiles the phage library enriched by panning on the pool of cancer sera. To further decrease the complexity of profiles we used computational methods for transforming a list of peptides constituting the mimotope profiles to the list motifs formed by similar peptide sequences. We have shown that the amino-acid order is meaningful in mimotope motifs since they contain significantly more peptides than motifs among peptides where amino-acids are randomly permuted. Also the single sample motifs significantly differ from motifs in peptides drawn from multiple samples. Finally, multiple cancer-specific motifs have been identified.

Novel ligands for cancer diagnosis: selection of peptide ligands for identification and isolation of B-cell lymphomas.

PubMed

McGuire, Michael J; Samli, Kausar N; Chang, Ya-Ching; Brown, Kathlynn C

2006-04-01

Lymphoma and leukemia account for nearly 8% of cancer fatalities each year. Present treatments do not differentiate between normal and malignant cells. New reagents that distinguish malignant cells and enable the isolation of these cells from the normal background will enhance the molecular characterization of disease and specificity of treatment. Peptide ligands were selected from a phage-displayed peptide library by biopanning on the B-cell lymphoma line, A20. The isolated peptides were assessed as reagents for identification and isolation of lymphoma cells by flow cytometry and cell capture with magnetic beads. Two novel peptides and one obtained previously on cardiomyocytes were selected. A20 cells bind phage displaying these peptides 250- to 450-fold over control phage. These phage bind to other bone marrow-derived cancel lines including some macrophage and T cells but do not bind to normal splenocytes. Synthetic constructs of these peptides have binding affinities comparable to B-cell-specific antibodies. Similar to antibodies, these peptides can be used in flow cytometry and magnetic bead capture to distinguish lymphoma cells from normal splenocytes. Bone marrow-derived malignant cells express cell surface markers that can be used to distinguish them from normal cells. These results demonstrate the ability to use an unbiased screen to rapidly generate high-affinity peptide ligands for identification and isolation of lymphoma cells.

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: peach Pru p 3 allergen as a model.

PubMed

Pacios, Luis F; Tordesillas, Leticia; Cuesta-Herranz, Javier; Compes, Esther; Sánchez-Monge, Rosa; Palacín, Arantxa; Salcedo, Gabriel; Díaz-Perales, Araceli

2008-04-01

Lipid transfer proteins (LTPs) are the major allergens of Rosaceae fruits in the Mediterranean area. Pru p 3, the LTP and major allergen of peach, is a suitable model for studying food allergy and amino acid sequences related with its IgE-binding capacity. In this work, we sought to map IgE mimotopes on the structure of Pru p 3, using the combination of a random peptide phage display library and a three-dimensional modelling approach. Pru p 3-specific IgE was purified from 2 different pools of sera from peach allergic patients grouped by symptoms (OAS-pool or SYS-pool), and used for screening of a random dodecapeptide phage display library. Positive clones were further confirmed by ELISA assays testing individual sera from each pool. Three-dimensional modelling allowed location of mimotopes based on analysis of electrostatic properties and solvent exposure of the Pru p 3 surface. Twenty-one phage clones were selected using Pru p 3-specific IgE, 9 of which were chosen using OAS-specific IgE while the other 12 were selected with systemic-specific IgE. Peptide alignments revealed consensus sequences for each pool: L37 R39 T40 P42 D43 R44 A46 P70 S76 P78 Y79 for OAS-IgE, and N35 N36 L37 R39 T40 D43 A46 S76 I77 P78 for systemic-IgE. These 2 consensus sequences were mapped on the same surface of Pru p 3, corresponding to the helix 2-loop-helix 3 region and part of the non-structured C-terminal coil. Thus, 2 relevant conformational IgE-binding regions of Pru p 3 were identified using a random peptide phage display library. Mimotopes can be used to study the interaction between allergens and IgE, and to accelerate the process to design new vaccines and new immunotherapy strategies.

Identification of Molecular Receptors for Therapeutic Targeting in Prostate Cancer

DTIC Science & Technology

2009-12-01

125:385–398. 11. Arap W, et al. (2002) Steps toward mapping the human vasculature by phage display. Nat Med 8:121–127. 12. Arap W, Pasqualini R...surface expression of the stress response chaperone GRP78 enables tumor targeting by circulating ligands. Cancer Cell 6:275–284. 14. Pasqualini R...Ruoslahti E (1996) Organ targeting in vivo using phage display peptide libraries. Nature 380:364–366. 15. Pasqualini R, Arap W, Rajotte D, Ruoslahti E (2001

Antibody phage display: overview of a powerful technology that has quickly translated to the clinic.

PubMed

Kotlan, Beatrix; Glassy, Mark C

2009-01-01

Antibody-based immunologic reagents are useful for identifying, isolating, or eliminating cells with particular characteristics related to different diseases. Phage display is a highly valuable technique for antibody selection related to this purpose. In brief, a diverse group of antibody genes prepared from a patient or generated in vitro are inserted into a phagemid vector or the phage genome so that when the protein is expressed, it becomes anchored on the surface of the phage by fusion to a coat protein. A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition. Phage that carry proteins or peptides bind preferentially to the target and can thus be isolated from the library. The viruses that are recovered in this way also carry the gene for the binding moiety facilitating its over-expression or manipulation. Recent reviews highlight key milestones in the development of antibody libraries and their screening by phage display, and the impact of these technologies on drug discovery seems assured.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-03-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

PubMed Central

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-01-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands. PMID:24670678

HIV-1 V3 loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features.

PubMed

Gazarian, Karlen G; Palacios-Rodríguez, Yadira; Gazarian, Tatiana G; Huerta, Leonor

2013-06-01

The crown region of the V3 loop in HIV-1 that contains the conserved amino acid sequence GPGR/G is known as the principal neutralizing determinant due to the extraordinary ability of antibodies to this region to neutralize the virus. To complement the existing peptide models of this epitope, we describe a family of 18 phage-displayed peptides, which include linear 12mer and constrained 7mer peptides that was selected by screening random libraries with serum from HIV-1 subtype B-infected patients. The 7mer constrained peptides presented two conserved amino acid sequences: PR-L in N-terminus and GPG in the C-terminus. On the basis of these peptides we propose a mimotope model of the V3 crown epitope in which the PR-L and GPG sequences represent the two known epitope binding sites. The GPG, has the same function as the V3 crown GPGR sequence but without the involvement of the "R" despite its being considered as the signature of the epitope in B-subtype viruses. The PR-L contains a proline not existing in the epitope that is postulated to induce kinks in the backbones of all peptides and create a spatial element mimicking the N-terminal conformationally variable binding site. Rabbit serum to these mimotopes recognized the V3 peptides and moderately decreased the fusion between HIV-1 Env- and CD4-expressing Jurkat cells. This study proposes the efficient generation by means of patient sera of V3 epitope mimics validated by interaction with the antibodies to contemporary viruses induced in patients. The serum antibody-selectable mimotopes are sources of novel information on the fine structure-function properties of HIV-1 principal neutralizing domain and candidate anti-HIV-1 immunogens. Copyright © 2012 Elsevier Ltd. All rights reserved.

Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy.

PubMed

Alban, Silvana Maria; de Moura, Juliana Ferreira; Thomaz-Soccol, Vanete; Bührer Sékula, Samira; Alvarenga, Larissa Magalhães; Mira, Marcelo Távora; Olortegui, Carlos Chávez; Minozzo, João Carlos

2014-01-01

The diagnosis of leprosy is primarily based on clinical manifestations, and there is no widely available laboratory test for the early detection of this disease, which is caused by Mycobacterium leprae. In fact, early detection and treatment are the key elements to the successful control of leprosy. Peptide ligands for antibodies from leprosy patients were selected from phage-displayed peptide libraries. Three peptide sequences expressed by reactive phage clones were chemically synthesized. Serological assays that used synthetic peptides were evaluated using serum samples from leprosy patients, household contacts (HC) of leprosy patients, tuberculosis patients and endemic controls (EC). A pool of three peptides identified 73.9% (17/23) of multibacillary (MB) leprosy patients using an enzyme-linked immunosorbent assay (ELISA). These peptides also showed some seroreactivities to the HC and EC individuals. The peptides were not reactive to rabbit polyclonal antisera against the different environmental mycobacteria. The same peptides that were conjugated to the carrier protein bovine serum albumin (BSA) induced the production of antibodies in the mice. The anti-peptide antibodies that were used in the Western blotting analysis of M. leprae crude extracts revealed a single band of approximately 30 kDa in one-dimensional electrophoresis and four 30 kDa isoforms in the two-dimensional gel. The Western blotting data indicated that the three peptides are derived from the same bacterial protein. These new antigens may be useful in the diagnosis of MB leprosy patients. Their potentials as diagnostic reagents must be more extensively evaluated in future studies using a large panel of positive and negative sera. Furthermore, other test approaches using peptides should be assessed to increase their sensitivity and specificity in detecting leprosy patients. We have revealed evidence in support of phage-displayed peptides as promising biotechnological tools for the design of leprosy diagnostic serological assays.

Crossing borders to bind proteins--a new concept in protein recognition based on the conjugation of small organic molecules or short peptides to polypeptides from a designed set.

PubMed

Baltzer, Lars

2011-06-01

A new concept for protein recognition and binding is highlighted. The conjugation of small organic molecules or short peptides to polypeptides from a designed set provides binder molecules that bind proteins with high affinities, and with selectivities that are equal to those of antibodies. The small organic molecules or peptides need to bind the protein targets but only with modest affinities and selectivities, because conjugation to the polypeptides results in molecules with dramatically improved binder performance. The polypeptides are selected from a set of only sixteen sequences designed to bind, in principle, any protein. The small number of polypeptides used to prepare high-affinity binders contrasts sharply with the huge libraries used in binder technologies based on selection or immunization. Also, unlike antibodies and engineered proteins, the polypeptides have unordered three-dimensional structures and adapt to the proteins to which they bind. Binder molecules for the C-reactive protein, human carbonic anhydrase II, acetylcholine esterase, thymidine kinase 1, phosphorylated proteins, the D-dimer, and a number of antibodies are used as examples to demonstrate that affinities are achieved that are higher than those of the small molecules or peptides by as much as four orders of magnitude. Evaluation by pull-down experiments and ELISA-based tests in human serum show selectivities to be equal to those of antibodies. Small organic molecules and peptides are readily available from pools of endogenous ligands, enzyme substrates, inhibitors or products, from screened small molecule libraries, from phage display, and from mRNA display. The technology is an alternative to established binder concepts for applications in drug development, diagnostics, medical imaging, and protein separation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Xiaoyong; Cai, Cuizan; Xiao, Fei

Highlights: • A specific aFGF-binding peptide AP8 was identified from a phage display library. • AP8 could inhibit aFGF-stimulated cell proliferation in a dose-dependent manner. • AP8 arrested the cell cycle at the G0/G1 phase by suppressing Cyclin D1. • AP8 could block the activation of Erk1/2 and Akt kinase. • AP8 counteracted proliferation and cell cycle via influencing PA2G4 and PCNA. - Abstract: It has been reported that acidic fibroblast growth factor (aFGF) is expressed in breast cancer and via interactions with fibroblast growth factor receptors (FGFRs) to promote the stage and grade of the disease. Thus, aFGF/FGFRs havemore » been considered essential targets in breast cancer therapy. We identified a specific aFGF-binding peptide (AGNWTPI, named AP8) from a phage display heptapeptide library with aFGF after four rounds of biopanning. The peptide AP8 contained two (TP) amino acids identical and showed high homology to the peptides of the 182–188 (GTPNPTL) site of high-affinity aFGF receptor FGFR1. Functional analyses indicated that AP8 specifically competed with the corresponding phage clone A8 for binding to aFGF. In addition, AP8 could inhibit aFGF-stimulated cell proliferation, arrested the cell cycle at the G0/G1 phase by increasing PA2G4 and suppressing Cyclin D1 and PCNA, and blocked the aFGF-induced activation of Erk1/2 and Akt kinase in both breast cancer cells and vascular endothelial cells. Therefore, these results indicate that peptide AP8, acting as an aFGF antagonist, is a promising therapeutic agent for the treatment of breast cancer.« less

Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents.

PubMed

Alban, Silvana M; de Moura, Juliana Ferreira; Minozzo, João Carlos; Mira, Marcelo Távora; Soccol, Vanete Thomaz

2013-01-25

An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.

Identification and immunogenicity of immunodominant mimotopes of outer membrane protein U (OmpU) of Vibrio mimicus from phage display peptide library.

PubMed

Cen, Junyu; Liu, Xueqin; Li, Jinnian; Zhang, Ming; Wang, Wei

2013-01-01

Vibrio mimicus (V. mimicus) is the causative agent of ascites disease in aquatic animals. Outer membrane protein U (OmpU) is an important antigen of V. mimicus, but its protective epitopes are still unclear. A random 12-mer phage-displayed peptide library was used to screen and identify immunodominant mimotopes of the OmpU protein in V. mimicus by panning against purified OmpU-specific polyclonal antibody. Then the immunogenicity and immunoprotection in fish of these mimotopes was evaluated. Nine positive phage clones presented seven different 12- peptide sequences and more than 50% of them carried a consensus core motif of DSSK-P. These positive clones reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by OmpU protein. Intraperitoneal injection of seven positive phage clones into fish induced a specific antibody response to OmpU protein. The fish immunized respectively with the positive phage clones C17, C24, C60 and C66 obtained 100% immunoprotective effect against experimental V. mimicus challenge. Taken together, these mimotopes presented by clone C17, C24, C60 and C66 were immunodominant mimotopes of the OmpU protein and exhibited a more appropriate candidate as epitope-based vaccine against V. mimicus infection in aquatic animals. Copyright © 2012 Elsevier Ltd. All rights reserved.

On the limited recognition of inorganic surfaces by short peptides compared with antibodies.

PubMed

Artzy-Schnirman, Arbel; Abu-Shah, Enas; Dishon, Matan; Soifer, Hadas; Sivan, Yotam; Reiter, Yoram; Benhar, Itai; Sivan, Uri

2014-06-01

The vast potential applications of biomolecules that bind inorganic surfaces led mostly to the isolation of short peptides that target selectively specific materials. The demonstrated differential affinity toward certain surfaces created the impression that the recognition capacity of short peptides may match that of rigid biomolecules. In the following, we challenge this view by comparing the capacity of antibody molecules to discriminate between the (100) and (111A) facets of a gallium arsenide semiconductor crystal with the capacity of short peptides to do the same. Applying selection from several peptide and single chain phage display libraries, we find a number of antibody molecules that bind preferentially a given crystal facet but fail to isolate, in dozens of attempts, a single peptide capable of such recognition. The experiments underscore the importance of rigidity to the recognition of inorganic flat targets and therefore set limitations on potential applications of short peptides in biomimetics. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Antiviral Peptides Targeting the West Nile Virus Envelope Protein▿

PubMed Central

Bai, Fengwei; Town, Terrence; Pradhan, Deepti; Cox, Jonathan; Ashish; Ledizet, Michel; Anderson, John F.; Flavell, Richard A.; Krueger, Joanna K.; Koski, Raymond A.; Fikrig, Erol

2007-01-01

West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses. PMID:17151121

Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

NASA Astrophysics Data System (ADS)

Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

2007-04-01

Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

Discovery of peptide drug carrier candidates for targeted multi-drug delivery into prostate cancer cells.

PubMed

Bashari, O; Redko, B; Cohen, A; Luboshits, G; Gellerman, G; Firer, M A

2017-11-01

Metastatic castration-resistant prostate cancer (mCRPC) remains essentially incurable. Targeted Drug Delivery (TDD) systems may overcome the limitations of current mCRPC therapies. We describe the use of strict criteria to isolate novel prostate cancer cell targeting peptides that specifically deliver drugs into target cells. Phage from a libraries displaying 7mer peptides were exposed to PC-3 cells and only internalized phage were recovered. The ability of these phage to internalize into other prostate cancer cells (LNCaP, DU-145) was validated. The displayed peptides of selected phage clones were synthesized and their specificity for target cells was validated in vitro and in vivo. One peptide (P12) which specifically targeted PC-3 tumors in vivo was incorporated into mono-drug (Chlorambucil, Combretastatin or Camptothecin) and dual-drug (Chlorambucil/Combretastatin or Chlorambucil/Camptothecin) PDCs and the cytotoxic efficacy of these conjugates for target cells was tested. Conjugation of P12 into dual-drug PDCs allowed discovery of new drug combinations with synergistic effects. The use of strict selection criteria can lead to discovery of novel peptides for use as drug carriers for TDD. PDCs represent an effective alternative to current modes of free drug chemotherapy for prostate cancer. Copyright © 2017. Published by Elsevier B.V.

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

Three polypeptides screened from phage display random peptide library may be the receptor polypeptide of Mycoplasma genitalium adhesion protein.

PubMed

Deng, Xiangying; Zhu, Youcong; Dai, Pei; Yu, Minjun; Chen, Liesong; Zhu, Cuiming; You, Xiaoxing; Li, Lingling; Zeng, Yanhua

2018-04-28

Mycoplasma genitalium adhesion protein (MgPa) is a major adhesin of M. genitalium, a human pathogen associated with a series of genitourinary tract diseases. MgPa plays a very important role in M. genitalium adhering to the host cells. However, the exact receptor peptides or proteins of MgPa are still poorly understood so far. Three polypeptides (V-H-W-D-F-R-Q-W-W-Q-P-S), (D-W-S-S-W-V -Y-R-D-P-Q-T) and (H-Y-I-D-F-R-W) were previously screened from a phage display random peptide library using recombinant MgPa (rMgPa) as a target molecule. In this study, three polypeptides were artificially synthesized and investigated as to whether they are potential receptors of MgPa. We found that rMgPa specifically bound to three synthesized polypeptides as determined via an indirect enzyme-linked immunosorbent assay (ELISA). Moreover, three polypeptides were further identified by indirect immunofluorescence microscopy (IFM). We confirmed that rMgPa and M. genitalium can adhere to SV-HUC-1 cells in vitro and that anti-rMgPa antibody and three synthesized polypeptides can partially inhibit the adherence of rMgPa and M. genitalium to SV-HUC-1 cells. In summary, these three polypeptides may be the essential receptor peptides of MgPa, and may aid in enhancing the understanding of biological function of MgPa and the possible pathogenic mechanism of M. genitalium. Copyright © 2018 Elsevier Ltd. All rights reserved.

A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity.

PubMed

Lindberg, Hanna; Härd, Torleif; Löfblom, John; Ståhl, Stefan

2015-09-01

The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide. © 2015 The Authors. Biotechnology Journal published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution-Non-Commercial-NoDerivs Licence, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components.

PubMed

San Segundo-Acosta, Pablo; Garranzo-Asensio, María; Oeo-Santos, Carmen; Montero-Calle, Ana; Quiralte, Joaquín; Cuesta-Herranz, Javier; Villalba, Mayte; Barderas, Rodrigo

2018-05-01

Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Humoral immune responses against gonadotropin releasing hormone elicited by immunization with phage-peptide constructs obtained via phage display.

PubMed

Samoylov, Alexandre; Cochran, Anna; Schemera, Bettina; Kutzler, Michelle; Donovan, Caitlin; Petrenko, Valery; Bartol, Frank; Samoylova, Tatiana

2015-12-20

Phage display is based on genetic engineering of phage coat proteins resulting in fusion peptides displayed on the surface of phage particles. The technology is widely used for generation of phages with novel characteristics for numerous applications in biomedicine and far beyond. The focus of this study was on development of phage-peptide constructs that stimulate production of antibodies against gonadotropin releasing hormone (GnRH). Phage-peptide constructs that elicit production of neutralizing GnRH antibodies can be used for anti-fertility and anti-cancer applications. Phage-GnRH constructs were generated via selection from a phage display library using several types of GnRH antibodies as selection targets. Such phage constructs were characterized for sequence similarities to GnRH peptide and frequency of their occurrence in the selection rounds. Five of the constructs with suitable characteristics were tested in mice as a single dose 5×10(11) virions (vir) vaccine and were found to be able to stimulate production of GnRH-specific antibodies, but not to suppress testosterone (indirect indicator of GnRH antibody neutralizing properties). Next, one of the constructs was tested at a higher dose of 2×10(12) vir per mouse in combination with a poly(lactide-co-glycolide) (PLGA)-based adjuvant. This resulted in multifold increase in GnRH antibody production and significant reduction of serum testosterone, indicating that antibodies produced in response to the phage-GnRH immunization possess neutralizing properties. To achieve optimal immune responses for desired applications, phage-GnRH constructs can be modified with respect to flanking sequences of GnRH-like peptides displayed on phage. Anticipated therapeutic effects also might be attained using optimized phage doses, a combination of several constructs in a single treatment, or application of adjuvants and advanced phage delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of Anti-Infectives Using Phage Display: Biological Agents against Bacteria, Viruses, and Parasites

PubMed Central

Huang, Johnny X.; Bishop-Hurley, Sharon L.

2012-01-01

The vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens. PMID:22664969

Development of anti-infectives using phage display: biological agents against bacteria, viruses, and parasites.

PubMed

Huang, Johnny X; Bishop-Hurley, Sharon L; Cooper, Matthew A

2012-09-01

The vast majority of anti-infective therapeutics on the market or in development are small molecules; however, there is now a nascent pipeline of biological agents in development. Until recently, phage display technologies were used mainly to produce monoclonal antibodies (MAbs) targeted against cancer or inflammatory disease targets. Patent disputes impeded broad use of these methods and contributed to the dearth of candidates in the clinic during the 1990s. Today, however, phage display is recognized as a powerful tool for selecting novel peptides and antibodies that can bind to a wide range of antigens, ranging from whole cells to proteins and lipid targets. In this review, we highlight research that exploits phage display technology as a means of discovering novel therapeutics against infectious diseases, with a focus on antimicrobial peptides and antibodies in clinical or preclinical development. We discuss the different strategies and methods used to derive, select, and develop anti-infectives from phage display libraries and then highlight case studies of drug candidates in the process of development and commercialization. Advances in screening, manufacturing, and humanization technologies now mean that phage display can make a significant contribution in the fight against clinically important pathogens.

Phage protein-targeted cancer nanomedicines

PubMed Central

Petrenko, V.A.; Jayanna, P.K.

2015-01-01

Nanoencapsulation of anticancer drugs improves their therapeutic indices by virtue of the enhanced permeation and retention effect which achieves passive targeting of nanoparticles in tumors. This effect can be significantly enhanced by active targeting of nanovehicles to tumors. Numerous ligands have been proposed and used in various studies with peptides being considered attractive alternatives to antibodies. This is further reinforced by the availability of peptide phage display libraries which offer an unlimited reservoir of target-specific probes. In particular landscape phages with multivalent display of target-specific peptides which enable the phage particle itself to become a nanoplatform creates a paradigm for high throughput selection of nanoprobes setting the stage for personalized cancer management. Despite its promise, this conjugate of combinatorial chemistry and nanotechnology has not made a significant clinical impact in cancer management due to a lack of using robust processes that facilitate scale-up and manufacturing. To this end we proposed the use of phage fusion protein as the navigating modules of novel targeted nanomedicine platforms which are described in this review. PMID:24269681

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Enriching peptide libraries for binding affinity and specificity through computationally directed library design

PubMed Central

Foight, Glenna Wink; Chen, T. Scott; Richman, Daniel; Keating, Amy E.

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model. PMID:28236241

Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

PubMed

Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

Molecular detection via hybrid peptide-semiconductor photonic devices

NASA Astrophysics Data System (ADS)

Estephan, E.; Saab, M.-b.; Martin, M.; Cloitre, T.; Larroque, C.; Cuisinier, F. J. G.; Malvezzi, A. M.; Gergely, C.

2011-03-01

The aim of this work was to investigate the possibilities to support device functionality that includes strongly confined and localized light emission and detection processes within nano/micro-structured semiconductors for biosensing applications. The interface between biological molecules and semiconductor surfaces, yet still under-explored is a key issue for improving biomolecular recognition in devices. We report on the use of adhesion peptides, elaborated via combinatorial phage-display libraries for controlled placement of biomolecules, leading to user-tailored hybrid photonic systems for molecular detection. An M13 bacteriophage library has been used to screen 1010 different peptides against various semiconductors to finally isolate specific peptides presenting a high binding capacity for the target surfaces. When used to functionalize porous silicon microcavities (PSiM) and GaAs/AlGaAs photonic crystals, we observe the formation of extremely thin (<1nm) peptide layers, hereby preserving the nanostructuration of the crystals. This is important to assure the photonic response of these tiny structures when they are functionalized by a biotinylated peptide layer and then used to capture streptavidin. Molecular detection was monitored via both linear and nonlinear optical measurements. Our linear reflectance spectra demonstrate an enhanced detection resolution via PSiM devices, when functionalized with the Si-specific peptide. Molecular capture at even lower concentrations (femtomols) is possible via the second harmonic generation of GaAs/AlGaAs photonic crystals when functionalized with GaAs-specific peptides. Our work demonstrates the outstanding value of adhesion peptides as interface linkers between semiconductors and biological molecules. They assure an enhanced molecular detection via both linear and nonlinear answers of photonic crystals.

Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis).

PubMed

Wu, Youjia; Wang, Lei; Zhou, Mei; Ma, Chengbang; Chen, Xiaole; Bai, Bing; Chen, Tianbao; Shaw, Chris

2011-06-01

Amphibian skin secretions are rich sources of biologically-active peptides with antimicrobial peptides predominating in many species. Several studies involving molecular cloning of biosynthetic precursor-encoding cDNAs from skin or skin secretions have revealed that these exhibit highly-conserved domain architectures with an unusually high degree of conserved nucleotide and resultant amino acid sequences within the signal peptides. This high degree of nucleotide sequence conservation has permitted the design of primers complementary to such sites facilitating "shotgun" cloning of skin or skin secretion-derived cDNA libraries from hitherto unstudied species. Here we have used such an approach using a skin secretion-derived cDNA library from an unstudied species of Chinese frog - the Fujian large-headed frog, Limnonectes fujianensis - and have discovered two 16-mer peptides of novel primary structures, named limnonectin-1Fa (SFPFFPPGICKRLKRC) and limnonectin-1Fb (SFHVFPPWMCKSLKKC), that represent the prototypes of a new class of amphibian skin antimicrobial peptide. Unusually these limnonectins display activity only against a Gram-negative bacterium (MICs of 35 and 70 μM) and are devoid of haemolytic activity at concentrations up to 160 μM. Thus the "shotgun" cloning approach described can exploit the unusually high degree of nucleotide conservation in signal peptide-encoding domains of amphibian defensive skin secretion peptide precursor-encoding cDNAs to rapidly expedite the discovery of novel and functional defensive peptides in a manner that circumvents specimen sacrifice without compromising robustness of data. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Targeting the atypical chemokine receptor ACKR3/CXCR7 for the treatment of cancer and other diseases

NASA Astrophysics Data System (ADS)

Vestal, Richard D., Jr.

One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's N-terminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, showed no effect on the viability or proliferation upon exposure to MCF-7 and U87-MG cells. Membrane binding, colocalization, and cellular uptake were confirmed by whole-cell ELISA and confocal microscopy. The recovered peptides did not activate the receptor as confirmed by a Beta-Arrestin recruitment assay. The data shows that the peptide sequences recovered from the phage display protocol are viable candidates for targeting cancer cells and delivering material to them.

Comprehensive mutational analysis of the M13 major coat protein: improved scaffolds for C-terminal phage display.

PubMed

Held, Heike A; Sidhu, Sachdev S

2004-07-09

A peptide was fused to the C terminus of the M13 bacteriophage major coat protein (P8), and libraries of P8 mutants were screened to select for variants that displayed the peptide with high efficiency. Over 600 variants were sequenced to compile a comprehensive database of P8 sequence diversity compatible with assembly into the wild-type phage coat. The database reveals that, while the alpha-helical P8 molecule was highly tolerant to mutations, certain functional epitopes were required for efficient incorporation. Three hydrophobic epitopes were located approximately equidistantly along the length of the alpha-helix. In addition, a positively charged epitope was required directly opposite the most C-terminal hydrophobic epitope and on the same side as the other two epitopes. Both ends of the protein were highly tolerant to mutations, consistent with the use of P8 as a scaffold for both N and C-terminal phage display. Further rounds of selection were used to enrich for P8 variants that supported higher levels of C-terminal peptide display. The largest improvements in display resulted from mutations around the junction between P8 and the C-terminal linker, and additional mutations in the N-terminal region were selected for further improvements in display. The best P8 variants improved C-terminal display more than 100-fold relative to the wild-type, and these variants could support the simultaneous display of N and C-terminal fusions. These finding provide information on the requirements for filamentous phage coat assembly, and provide improved scaffolds for phage display technology. Copyright 2004 Elsevier Ltd.

Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule.

PubMed

Takakusagi, Yoichi; Takakusagi, Kaori; Sugawara, Fumio; Sakaguchi, Kengo

2018-01-01

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.

Discovery of human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) immunocontraceptive epitopes and their effects on fertility in male and female mice.

PubMed

Chen, Xuemei; Liu, Xiaodong; Ren, Xiuhua; Li, Xuewu; Wang, Li; Zang, Weidong

2016-03-01

The key goals of immunocontraception research are to obtain full contraceptive effects using vaccines administered to both males and females. Current research concerning human anti-sperm contraceptive vaccines is focused on delineating infertility-related epitopes to avoid autoimmune disease. We constructed phage-display peptide libraries to select epitope peptides derived from human posterior head 20 (hPH20) and homo sapiens sperm acrosome associated 1 (hSPACA1) using sera collected from infertile women harbouring anti-sperm antibodies. Following five rounds of selection, positive colonies were reconfirmed for reactivity with the immunoinfertile sera. We biopanned and analysed the chemical properties of four epitope peptides, named P82, Sa6, Sa37 and Sa76. Synthetic peptides were made and coupled to either bovine serum albumin (BSA) or ovalbumin. We used the BSA-conjugated peptides to immunise BALB/c mice and examined the effects on fertility in female and male mice. The synthetic peptides generated a sperm-specific antibody response in female and male mice that caused a contraceptive state. The immunocontraceptive effect was reversible and, with the disappearance of peptide-specific antibodies, there was complete restoration of fertility. Vaccinations using P82, Sa6 and Sa76 peptides resulted in no apparent side effects. Thus, it is efficient and practical to identify epitope peptide candidates by phage display. These peptides may find clinical application in the specific diagnosis and treatment of male and female infertility and contraceptive vaccine development.

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

PSBinder: A Web Service for Predicting Polystyrene Surface-Binding Peptides.

PubMed

Li, Ning; Kang, Juanjuan; Jiang, Lixu; He, Bifang; Lin, Hao; Huang, Jian

2017-01-01

Polystyrene surface-binding peptides (PSBPs) are useful as affinity tags to build a highly effective ELISA system. However, they are also a quite common type of target-unrelated peptides (TUPs) in the panning of phage-displayed random peptide library. As TUP, PSBP will mislead the analysis of panning results if not identified. Therefore, it is necessary to find a way to quickly and easily foretell if a peptide is likely to be a PSBP or not. In this paper, we describe PSBinder, a predictor based on SVM. To our knowledge, it is the first web server for predicting PSBP. The SVM model was built with the feature of optimized dipeptide composition and 87.02% (MCC = 0.74; AUC = 0.91) of peptides were correctly classified by fivefold cross-validation. PSBinder can be used to exclude highly possible PSBP from biopanning results or to find novel candidates for polystyrene affinity tags. Either way, it is valuable for biotechnology community.

Binding of leachable components of polymethyl methacrylate (PMMA) and peptide on modified SPR chip

NASA Astrophysics Data System (ADS)

Szaloki, M.; Vitalyos, G.; Harfalvi, J.; Hegedus, Cs

2013-12-01

Many types of polymers are often used in dentistry, which may cause allergic reaction, mainly methyl methacrylate allergy due to the leachable, degradable components of polymerized dental products. The aim of this study was to investigate the interaction between the leachable components of PMMA and peptides by Fourier-transform Surface Plasmon Resonance (FT SPR). In our previous work binding of oligopeptides (Ph.D.-7 and Ph.D.-12 Peptide Library Kit) was investigated to PMMA surface by phage display technique. It was found that oligopeptides bounded specifically to PMMA surface. The most common amino acids were leucine and proline inside the amino acids sequences of DNA of phages. The binding of haptens, as formaldehyde and methacrylic acid, to frequent amino acids was to investigate on the modified gold SPR chip. Self assembled monolayer (SAM) modified the surface of gold chip and ensured the specific binding between the haptens and amino acids. It was found that amino acids bounded to modified SPR gold and the haptens bounded to amino acids by creating multilayer on the chip surface. By the application of phage display and SPR modern bioanalytical methods the interaction between allergens and peptides can be investigated.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

PubMed

Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P

2016-07-01

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

PubMed Central

Wu, Jemma X.; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.

2016-01-01

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

PubMed

Khalili, Ehsan; Lakzaei, Mostafa; Rasaee, Mohhamad Javad; Aminian, Mahdi

2015-10-01

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

Peptides for functionalization of InP semiconductors.

PubMed

Estephan, Elias; Saab, Marie-belle; Larroque, Christian; Martin, Marta; Olsson, Fredrik; Lourdudoss, Sebastian; Gergely, Csilla

2009-09-15

The challenge is to achieve high specificity in molecular sensing by proper functionalization of micro/nano-structured semiconductors by peptides that reveal specific recognition for these structures. Here we report on surface modification of the InP semiconductors by adhesion peptides produced by the phage display technique. An M13 bacteriophage library has been used to screen 10(10) different peptides against the InP(001) and the InP(111) surfaces to finally isolate specific peptides for each orientation of the InP. MALDI-TOF/TOF mass spectrometry has been employed to study real affinity of the peptide towards the InP surfaces. The peptides serve for controlled placement of biotin onto InP to bind then streptavidin. Our Atomic Force Microscopy study revealed a total surface coverage of molecules when the InP surface was functionalized by its specific biotinylated peptide (YAIKGPSHFRPS). Finally, fluorescence microscopy has been employed to demonstrate the preferential attachment of the peptide onto a micro-patterned InP surface. Use of substrate specific peptides could present an alternative solution for the problems encountered in the actually existing sensing methods and molecular self-assembly due to the unwanted unspecific interactions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wenhui; Chen, Xilei; Li, Tao

Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163–169 (LLAVPAA) directly participating in ligandmore » binding, carried the same charges as its corresponding motif (aa163–169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer. - Highlights: ► A novel FGF8b-binding peptide P12 was isolated from a phage display library. ► The mechanisms for P12 peptide inhibiting cell proliferation were proposed. ► P12 caused cell cycle arrest at G0/G1 phase via suppression of Cyclin D1 and PCNA. ► P12 suppressed FGF8b-induced activations of Akt and MAP kinases. ► P12 acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.« less

Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

PubMed

Huang, Renhua; Fang, Pete; Kay, Brian K

2012-09-01

Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

PubMed Central

Matochko, Wadim L.; Derda, Ratmir

2013-01-01

Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N × 1 frequency vector n = ||ni||, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N × N matrix and a stochastic sampling operator (S a). The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of S a and use them to define the sequencing operator (S e q). Sequencing without any bias and errors is S e q = S a IN, where IN is a N × N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (C E N), which describes elimination or statistically significant downsampling, of specific reads during the sequencing process. PMID:24416071

Mixture-based combinatorial libraries from small individual peptide libraries: a case study on α1-antitrypsin deficiency.

PubMed

Chang, Yi-Pin; Chu, Yen-Ho

2014-05-16

The design, synthesis and screening of diversity-oriented peptide libraries using a "libraries from libraries" strategy for the development of inhibitors of α1-antitrypsin deficiency are described. The major buttress of the biochemical approach presented here is the use of well-established solid-phase split-and-mix method for the generation of mixture-based libraries. The combinatorial technique iterative deconvolution was employed for library screening. While molecular diversity is the general consideration of combinatorial libraries, exquisite design through systematic screening of small individual libraries is a prerequisite for effective library screening and can avoid potential problems in some cases. This review will also illustrate how large peptide libraries were designed, as well as how a conformation-sensitive assay was developed based on the mechanism of the conformational disease. Finally, the combinatorially selected peptide inhibitor capable of blocking abnormal protein aggregation will be characterized by biophysical, cellular and computational methods.

Functional display of platelet-binding VWF fragments on filamentous bacteriophage.

PubMed

Yee, Andrew; Tan, Fen-Lai; Ginsburg, David

2013-01-01

von Willebrand factor (VWF) tethers platelets to sites of vascular injury via interaction with the platelet surface receptor, GPIb. To further define the VWF sequences required for VWF-platelet interaction, a phage library displaying random VWF protein fragments was screened against formalin-fixed platelets. After 3 rounds of affinity selection, DNA sequencing of platelet-bound clones identified VWF peptides mapping exclusively to the A1 domain. Aligning these sequences defined a minimal, overlapping segment spanning P1254-A1461, which encompasses the C1272-C1458 cystine loop. Analysis of phage carrying a mutated A1 segment (C1272/1458A) confirmed the requirement of the cystine loop for optimal binding. Four rounds of affinity maturation of a randomly mutagenized A1 phage library identified 10 and 14 unique mutants associated with enhanced platelet binding in the presence and absence of botrocetin, respectively, with 2 mutants (S1370G and I1372V) common to both conditions. These results demonstrate the utility of filamentous phage for studying VWF protein structure-function and identify a minimal, contiguous peptide that bind to formalin-fixed platelets, confirming the importance of the VWF A1 domain with no evidence for another independently platelet-binding segment within VWF. These findings also point to key structural elements within the A1 domain that regulate VWF-platelet adhesion.

Novel Synthetic Antimicrobial Peptides against Streptococcus mutans▿

PubMed Central

He, Jian; Eckert, Randal; Pharm, Thanh; Simanian, Maurice D.; Hu, Chuhong; Yarbrough, Daniel K.; Qi, Fengxia; Anderson, Maxwell H.; Shi, Wenyuan

2007-01-01

Streptococcus mutans, a common oral pathogen and the causative agent of dental caries, has persisted and even thrived on the tooth surface despite constant removal and eradication efforts. In this study, we generated a number of synthetic antimicrobial peptides against this bacterium via construction and screening of several structurally diverse peptide libraries where the hydrophobicity and charge within each library was varied incrementally in order to generate a collection of peptides with different biochemical characteristics. From these libraries, we identified multiple peptides with robust killing activity against S. mutans. To further improve their effectiveness, the most bactericidal peptides from each library were synthesized together as one molecule, in various combinations, with and without a flexible peptide linker between each antimicrobial region. Many of these “fusion” peptides had enhanced killing activities in comparison with those of the original nonconjoined molecules. The results presented here illustrate that small libraries of biochemically constrained peptides can be used to generate antimicrobial peptides against S. mutans, several of which may be likely candidates for the development of anticaries agents. PMID:17296741

CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

PubMed

Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

2011-04-25

We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

PubMed

Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

2017-11-13

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Stable-isotope-labeled Histone Peptide Library for Histone Post-translational Modification and Variant Quantification by Mass Spectrometry *

PubMed Central

Lin, Shu; Wein, Samuel; Gonzales-Cope, Michelle; Otte, Gabriel L.; Yuan, Zuo-Fei; Afjehi-Sadat, Leila; Maile, Tobias; Berger, Shelley L.; Rush, John; Lill, Jennie R.; Arnott, David; Garcia, Benjamin A.

2014-01-01

To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields. PMID:25000943

Mapping of epitopes for autoantibodies to the type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modeling: overlap of antibody and T cell determinants.

PubMed

Dromey, James A; Weenink, Sarah M; Peters, Günther H; Endl, Josef; Tighe, Patrick J; Todd, Ian; Christie, Michael R

2004-04-01

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.

Rapid development of new protein biosensors utilizing peptides obtained via phage display.

PubMed

Wu, Jun; Park, Jong Pil; Dooley, Kevin; Cropek, Donald M; West, Alan C; Banta, Scott

2011-01-01

There is a consistent demand for new biosensors for the detection of protein targets, and a systematic method for the rapid development of new sensors is needed. Here we present a platform where short unstructured peptides that bind to a desired target are selected using M13 phage display. The selected peptides are then chemically synthesized and immobilized on gold, allowing for detection of the target using electrochemical techniques such as electrochemical impedance spectroscopy (EIS). A quartz crystal microbalance (QCM) is also used as a diagnostic tool during biosensor development. We demonstrate the utility of this approach by creating a novel peptide-based electrochemical biosensor for the enzyme alanine aminotransferase (ALT), a well-known biomarker of hepatotoxicity. Biopanning of the M13 phage display library over immobilized ALT, led to the rapid identification of a new peptide (ALT5-8) with an amino acid sequence of WHWRNPDFWYLK. Phage particles expressing this peptide exhibited nanomolar affinity for immobilized ALT (K(d,app) = 85±20 nM). The newly identified ALT5-8 peptide was then chemically synthesized with a C-terminal cysteine for gold immobilization. The performance of the gold-immobilized peptides was studied with cyclic voltammetry (CV), QCM, and EIS. Using QCM, the sensitivity for ALT detection was 8.9±0.9 Hz/(µg/mL) and the limit of detection (LOD) was 60 ng/mL. Using EIS measurements, the sensitivity was 142±12 impedance percentage change %/(µg/mL) and the LOD was 92 ng/mL. In both cases, the LOD was below the typical concentration of ALT in human blood. Although both QCM and EIS produced similar LODs, EIS is preferable due to a larger linear dynamic range. Using QCM, the immobilized peptide exhibited a nanomolar dissociation constant for ALT (K(d) = 20.1±0.6 nM). These results demonstrate a simple and rapid platform for developing and assessing the performance of sensitive, peptide-based biosensors for new protein targets.

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

PubMed Central

Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

2006-01-01

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

Mimtags: the use of phage display technology to produce novel protein-specific probes.

PubMed

Ahmed, Nayyar; Dhanapala, Pathum; Sadli, Nadia; Barrow, Colin J; Suphioglu, Cenk

2014-03-01

In recent times the use of protein-specific probes in the field of proteomics has undergone evolutionary changes leading to the discovery of new probing techniques. Protein-specific probes serve two main purposes: epitope mapping and detection assays. One such technique is the use of phage display in the random selection of peptide mimotopes (mimtags) that can tag epitopes of proteins, replacing the use of monoclonal antibodies in detection systems. In this study, phage display technology was used to screen a random peptide library with a biologically active purified human interleukin-4 receptor (IL-4R) and interleukin-13 (IL-13) to identify mimtag candidates that interacted with these proteins. Once identified, the mimtags were commercially synthesised, biotinylated and used for in vitro immunoassays. We have used phage display to identify M13 phage clones that demonstrated specific binding to IL-4R and IL-13 cytokine. A consensus in binding sequences was observed and phage clones characterised had identical peptide sequence motifs. Only one was synthesised for use in further immunoassays, demonstrating significant binding to either IL-4R or IL-13. We have successfully shown the use of phage display to identify and characterise mimtags that specifically bind to their target epitope. Thus, this new method of probing proteins can be used in the future as a novel tool for immunoassay and detection technique, which is cheaper and more rapidly produced and therefore a better alternative to the use of monoclonal antibodies. Copyright © 2014 Elsevier B.V. All rights reserved.

An In Vitro Translation, Selection, and Amplification System for Peptide Nucleic Acids

PubMed Central

Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.

2009-01-01

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory. PMID:20081830

Encoded libraries of chemically modified peptides.

PubMed

Heinis, Christian; Winter, Greg

2015-06-01

The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries. Copyright © 2015. Published by Elsevier Ltd.

Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU.

PubMed

Hoff, Kevin G; Ta, Dennis T; Tapley, Tim L; Silberg, Jonathan J; Vickery, Larry E

2002-07-26

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.

Identification of Molecular Receptors for Therapeutic Targeting in Prostate Cancer

DTIC Science & Technology

2006-12-01

prostate cancer: potential role of androgen and ErbB receptor signal transduction crosstalk. Neoplasia. 5, 99-109 (2003). 10. Kolonin, M., Pasqualini , R...and Arap, W. Molecular addresses in blood vessels as targets for therapy. Curr. Opin. Chem. Biol. 5, 308-313 (2001). 11. Pasqualini , R., and...Ruoslahti, E. Organ targeting in vivo using phage display peptide libraries. Nature 380, 364-366 (1996). 12. Arap, W., Pasqualini , R., and Ruoslahti, E

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

PubMed

Hiemstra, H S; van Veelen, P A; Schloot, N C; Geluk, A; van Meijgaarden, K E; Willemen, S J; Leunissen, J A; Benckhuijsen, W E; Amons, R; de Vries, R R; Roep, B O; Ottenhoff, T H; Drijfhout, J W

1998-10-15

Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

Libraries of Peptide Fragmentation Mass Spectra Database

National Institute of Standards and Technology Data Gateway

SRD 1C NIST Libraries of Peptide Fragmentation Mass Spectra Database (Web, free access)   The purpose of the library is to provide peptide reference data for laboratories employing mass spectrometry-based proteomics methods for protein analysis. Mass spectral libraries identify these compounds in a more sensitive and robust manner than alternative methods. These databases are freely available for testing and development of new applications.

Subtractive phage display selection for screening and identification of peptide sequences with potential use in serodiagnosis of paracoccidioidomycosis caused by Paracoccidioides brasiliensis.

PubMed

Portes, L da Silva; Kioshima, E S; de Camargo, Z P; Batista, W L; Xander, P

2017-11-01

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease endemic in Latin America whose aetiologic agents are the thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. Despite technological advances, some problems have been reported for the fungal antigens used for serological diagnosis, and inconsistencies among laboratories have been reported. The use of synthetic peptides in the serological diagnosis of infectious diseases has proved to be a valuable strategy because in some cases, the reactions are more specific and sensitive. In this study, we used a subtractive selection with a phage display library against purified polyclonal antibodies for negative and positive PCM sera caused by P. brasiliensis. The binding phages were sequenced and tested in a binding assay to evaluate its interaction with sera from normal individuals and PCM patients. Synthetic peptides derived from these phage clones were tested in a serological assay, and we observed a significant recognition of LP15 by sera from PCM patients infected with P. brasiliensis. Our results demonstrated that subtractive phage display selection may be useful for identifying new epitopes that can be applied to the serodiagnosis of PCM caused by P. brasiliensis. Currently, there is no standardized method for the preparation of paracoccidioidomycosis (PCM) antigens, which has resulted in differences in the antigens used for serological diagnosis. Here, we report a procedure that uses subtractive phage display selection to select and identify new epitopes for the serodiagnosis of PCM caused by Paracoccidioides brasiliensis. A synthetic peptide obtained using this methodology was successfully recognized by sera from PCM patients, thus demonstrating its potential use for improving the serodiagnosis of this mycosis. The development of synthetic peptides for the serodiagnosis of PCM could be a promising alternative for the better standardization of diagnoses among laboratories. © 2017 The Society for Applied Microbiology.

Phage display—A powerful technique for immunotherapy

PubMed Central

Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

2012-01-01

One of the most effective molecular diversity techniques is phage display. This technology is based on a direct linkage between phage phenotype and its encapsulated genotype, which leads to presentation of molecule libraries on the phage surface. Phage display is utilized in studying protein-ligand interactions, receptor binding sites and in improving or modifying the affinity of proteins for their binding partners. Generating monoclonal antibodies and improving their affinity, cloning antibodies from unstable hybridoma cells and identifying epitopes, mimotopes and functional or accessible sites from antigens are also important advantages of this technology. Techniques originating from phage display have been applied to transfusion medicine, neurological disorders, mapping vascular addresses and tissue homing of peptides. Phages have been applicable to immunization therapies, which may lead to development of new tools used for treating autoimmune and cancer diseases. This review describes the phage display technology and presents the recent advancements in therapeutic applications of phage display. PMID:22906939

Effects of 8-mer acidic peptide concentration on the morphology and photoluminescence of synthesized ZnO nanomaterials

NASA Astrophysics Data System (ADS)

Moon, Chung Hee; Tousi, Marzieh; Cheeney, Joseph; Ngo-Duc, Tam-Triet; Zuo, Zheng; Liu, Jianlin; Haberer, Elaine D.

2015-11-01

An 8-mer ZnO-binding peptide, VPGAAEHT, was identified using a M13 pVIII phage display library and employed as an additive during aqueous-based ZnO synthesis at 65 °C. Unlike most other well-studied ZnO-binding sequences which are strongly basic (pI > pH 7), the 8-mer peptide was overall acidic (pI < pH 7) in character, including only a single basic residue. The selected peptide strongly influenced ZnO nanostructure formation. Morphology and optical emission properties were found to be dependent on the concentration of peptide additive. Using lower peptide concentrations (<0.1 mM), single crystal hexagonal rods and platelets were produced, and using higher peptide concentrations (≥0.1 mM), polycrystalline layered platelets, yarn-like structures, and microspheres were assembled. Photoluminescence analysis revealed a characteristic ZnO band-edge peak, as well as sub-bandgap emission peaks. Defect-related green emission, typically associated with surface-related oxygen and zinc vacancies, was significantly reduced by the peptide additive, while blue emission, attributable to oxygen and zinc interstitials, emerged with increased peptide concentrations. Peptide-directed synthesis of ZnO materials may be useful for gas sensing and photocatalytic applications in which properly engineered morphology and defect levels have demonstrated enhanced performance.

Comprehensive peptidomimetic libraries targeting protein-protein interactions.

PubMed

Whitby, Landon R; Boger, Dale L

2012-10-16

Transient protein-protein interactions (PPIs) are essential components in cellular signaling pathways as well as in important processes such as viral infection, replication, and immune suppression. The unknown or uncharacterized PPIs involved in such interaction networks often represent compelling therapeutic targets for drug discovery. To date, however, the main strategies for discovery of small molecule modulators of PPIs are typically limited to structurally characterized targets. Recent developments in molecular scaffolds that mimic the side chain display of peptide secondary structures have yielded effective designs, but few screening libraries of such mimetics are available to interrogate PPI targets. We initiated a program to prepare a comprehensive small molecule library designed to mimic the three major recognition motifs that mediate PPIs (α-helix, β-turn, and β-strand). Three libraries would be built around templates designed to mimic each such secondary structure and substituted with all triplet combinations of groups representing the 20 natural amino acid side chains. When combined, the three libraries would contain a member capable of mimicking the key interaction and recognition residues of most targetable PPIs. In this Account, we summarize the results of the design, synthesis, and validation of an 8000 member α-helix mimetic library and a 4200 member β-turn mimetic library. We expect that the screening of these libraries will not only provide lead structures against α-helix- or β-turn-mediated protein-protein or peptide-receptor interactions, even if the nature of the interaction is unknown, but also yield key insights into the recognition motif (α-helix or β-turn) and identify the key residues mediating the interaction. Consistent with this expectation, the screening of the libraries against p53/MDM2 and HIV-1 gp41 (α-helix mimetic library) or the opioid receptors (β-turn mimetic library) led to the discovery of library members expected to mimic the known endogenous ligands. These efforts led to the discovery of high-affinity α-helix mimetics (K(i) = 0.7 μM) against HIV-1 gp41 as well as high-affinity and selective β-turn mimetics (K(i) = 80 nM) against the κ-opioid receptor. The results suggest that the use of such comprehensive libraries of peptide secondary structure mimetics, built around effective molecular scaffolds, constitutes a powerful method of interrogating PPIs. These structures provide small molecule modulators of PPI networks for therapeutic target validation, lead compound discovery, and the identification of modulators of biological processes for further study.

[A novel protein equalizer based on single chain variable fragment display M13 phage library for nephropathy patient urine study].

PubMed

Zhao, Peng; Tao, Dingyin; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2009-05-01

A novel protein equalizer was developed with single chain variable fragment (scFv) library displaying M13 phage covalently bonded on monolithic cryogel. Due to the great number and various kinds of displayed scFv fragments, as well as strong and specific binding capacity between scFv fragments and proteins, a new protein equalizer technology is preferable in the pretreatment of complex protein samples. After the sample dissolved in phosphate buffer solution (PBS), it was repeatedly loaded onto the equalizer for five times, the bound proteins were in sequence eluted by 2 mol/L NaCl and 50 mmol/L Gly-HC1 (pH 2.5) solution, followed by digestion with thrombin. All proteins or peptides collected from each fraction were further analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (RPLC-ESI-MS/MS) with a serially coupled long microcolumn. Compared with the untreated samples, the identified protein number was increased from 142 to 396. Furthermore, from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results, it was found that the protein concentration difference was reduced obviously in the eluant of direct sample loading, and most high abundance proteins were identified in the eluant of NaCl. All these results demonstrate that the novel protein equalizer with scFv display M13 phage library immobilized on cyrogel could effectively reduce the dynamic range of proteins in complex samples, enabling the identification of more low abundance proteins.

Spectrum-to-Spectrum Searching Using a Proteome-wide Spectral Library*

PubMed Central

Yen, Chia-Yu; Houel, Stephane; Ahn, Natalie G.; Old, William M.

2011-01-01

The unambiguous assignment of tandem mass spectra (MS/MS) to peptide sequences remains a key unsolved problem in proteomics. Spectral library search strategies have emerged as a promising alternative for peptide identification, in which MS/MS spectra are directly compared against a reference library of confidently assigned spectra. Two problems relate to library size. First, reference spectral libraries are limited to rediscovery of previously identified peptides and are not applicable to new peptides, because of their incomplete coverage of the human proteome. Second, problems arise when searching a spectral library the size of the entire human proteome. We observed that traditional dot product scoring methods do not scale well with spectral library size, showing reduction in sensitivity when library size is increased. We show that this problem can be addressed by optimizing scoring metrics for spectrum-to-spectrum searches with large spectral libraries. MS/MS spectra for the 1.3 million predicted tryptic peptides in the human proteome are simulated using a kinetic fragmentation model (MassAnalyzer version2.1) to create a proteome-wide simulated spectral library. Searches of the simulated library increase MS/MS assignments by 24% compared with Mascot, when using probabilistic and rank based scoring methods. The proteome-wide coverage of the simulated library leads to 11% increase in unique peptide assignments, compared with parallel searches of a reference spectral library. Further improvement is attained when reference spectra and simulated spectra are combined into a hybrid spectral library, yielding 52% increased MS/MS assignments compared with Mascot searches. Our study demonstrates the advantages of using probabilistic and rank based scores to improve performance of spectrum-to-spectrum search strategies. PMID:21532008

Rapid Generation of a Nanocrystal-Labeled Peptide Library for Specific Identification of the Bacterium Clostrium Botulinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tok, J B

2004-11-11

Several peptide libraries containing up to 2 million unique peptide ligands have been synthesized. The peptides are attached onto a 80 micron resin and the length of these peptide ligands ranges from 5 to 9 amino acid residues. Using a novel calorimetric assay, the libraries were screened for binding to the ganglioside-binding domain of Clostridium Tetanus Toxin, a structural similar analog of the Clostridium Botulinum toxin. Several binding peptide sequences were identified, in which the detailed binding kinetics are currently underway using the Surface Plasmon Resonance (SPR) technique.

Engineering emergent multicellular behavior through synthetic adhesion

NASA Astrophysics Data System (ADS)

Glass, David; Riedel-Kruse, Ingmar

In over a decade, synthetic biology has developed increasingly robust gene networks within single cells, but constructed very few systems that demonstrate multicellular spatio-temporal dynamics. We are filling this gap in synthetic biology's toolbox by developing an E. coli self-assembly platform based on modular cell-cell adhesion. We developed a system in which adhesive selectivity is provided by a library of outer membrane-displayed peptides with intra-library specificities, while affinity is provided by consistent expression across the entire library. We further provide a biophysical model to help understand the parameter regimes in which this tool can be used to self-assemble into cellular clusters, filaments, or meshes. The combined platform will enable future development of synthetic multicellular systems for use in consortia-based metabolic engineering, in living materials, and in controlled study of minimal multicellular systems. Stanford Bio-X Bowes Fellowship.

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

PECAN: Library Free Peptide Detection for Data-Independent Acquisition Tandem Mass Spectrometry Data

PubMed Central

Ting, Ying S.; Egertson, Jarrett D.; Bollinger, James G.; Searle, Brian C.; Payne, Samuel H.; Noble, William Stafford; MacCoss, Michael J.

2017-01-01

In mass spectrometry-based shogun proteomics, data-independent acquisition (DIA) is an emerging technique for unbiased and reproducible measurement of protein mixtures. Without targeting a specific precursor ion, DIA MS/MS spectra are often highly multiplexed, containing product ions from multiple co-fragmenting precursors. Thus, detecting peptides directly from DIA data is challenging; most DIA data analyses require spectral libraries. Here we present a new library-free, peptide-centric tool PECAN that detects peptides directly from DIA data. PECAN reports evidence of detection based on product ion scoring, enabling detection of low abundance analytes with poor precursor ion signal. We benchmarked PECAN with chromatographic peak picking accuracy and peptide detection capability. We further validated PECAN detection with data-dependent acquisition and targeted analyses. Last, we used PECAN to build a library from DIA data and to query sequence variants. Together, these results show that PECAN detects peptides robustly and accurately from DIA data without using a library. PMID:28783153

A large synthetic peptide and phosphopeptide reference library for mass spectrometry-based proteomics.

PubMed

Marx, Harald; Lemeer, Simone; Schliep, Jan Erik; Matheron, Lucrece; Mohammed, Shabaz; Cox, Jürgen; Mann, Matthias; Heck, Albert J R; Kuster, Bernhard

2013-06-01

We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.

Proven in vitro evolution of protease cathepsin E-inhibitors and -activators at pH 4.5 using a paired peptide method.

PubMed

Kitamura, Koichiro; Komatsu, Masayuki; Biyani, Madhu; Futakami, Masae; Kawakubo, Tomoyo; Yamamoto, Kenji; Nishigaki, Koichi

2012-12-01

Improving a particular function of molecules is often more difficult than identifying such molecules ab initio. Here, a method to acquire higher affinity and/or more functional peptides was developed as a progressive library selection method. The primary library selection products were utilized to build a secondary library composed of blocks of 4 amino acids, of which selection led to peptides with increased activity. These peptides were further converted to randomly generate paired peptides. Cathepsin E-inhibitors thus obtained exhibited the highest activities and affinities (pM order). This was also the case with cathepsin E-activating peptides, proving the methodological effectiveness. The primary, secondary, and tertiary library selections can be regarded as module-finding, module-shuffling, and module-pairing, respectively, which resembles the progression of the natural evolution of proteins. The mode of peptide binding to their target proteins is discussed in analogy to antibodies and epitopes of an antigen. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Mapping a disordered portion of the Brz2001-binding site on a plant monooxygenase, DWARF4, using a quartz-crystal microbalance biosensor-based T7 phage display.

PubMed

Takakusagi, Yoichi; Manita, Daisuke; Kusayanagi, Tomoe; Izaguirre-Carbonell, Jesus; Takakusagi, Kaori; Kuramochi, Kouji; Iwabata, Kazuki; Kanai, Yoshihiro; Sakaguchi, Kengo; Sugawara, Fumio

2013-04-01

In small-molecule/protein interaction studies, technical difficulties such as low solubility of small molecules or low abundance of protein samples often restrict the progress of research. Here, we describe a quartz-crystal microbalance (QCM) biosensor-based T7 phage display in combination use with a receptor-ligand contacts (RELIC) bioinformatics server for application in a plant Brz2001/DWARF4 system. Brz2001 is a brassinosteroid biosynthesis inhibitor in the less-soluble triazole series of compounds that targets DWARF4, a cytochrome P450 (Cyp450) monooxygenase containing heme and iron. Using a Brz2001 derivative that has higher solubility in 70% EtOH and forms a self-assembled monolayer on gold electrode, we selected 34 Brz2001-recognizing peptides from a 15-mer T7 phage-displayed random peptide library using a total of four sets of one-cycle biopanning. The RELIC/MOTIF program revealed continuous and discontinuous short motifs conserved within the 34 Brz2001-selected 15-mer peptide sequences, indicating the increase of information content for Brz2001 recognition. Furthermore, an analysis of similarity between the 34 peptides and the amino-acid sequence of DWARF4 using the RELIC/MATCH program generated a similarity plot and a cluster diagram of the amino-acid sequence. Both of these data highlighted an internally located disordered portion of a catalytic site on DWARF4, indicating that this portion is essential for Brz2001 recognition. A similar trend was also noted by an analysis using another 26 Brz2001-selected peptides, and not observed using the 27 gold electrode-recognizing control peptides, demonstrating the reproducibility and specificity of this method. Thus, this affinity-based strategy enables high-throughput detection of the small-molecule-recognizing portion on the target protein, which overcomes technical difficulties such as sample solubility or preparation that occur when conventional methods are used.

In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses ▿ †

PubMed Central

Grimm, Dirk; Lee, Joyce S.; Wang, Lora; Desai, Tushar; Akache, Bassel; Storm, Theresa A.; Kay, Mark A.

2008-01-01

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications. PMID:18400866

Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

PubMed

Rhaiem, Rafik Ben; Houimel, Mehdi

2016-07-01

Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Hao; Dranchak, Patricia; Li, Zhiru

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanismmore » placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small-molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.« less

Peptide library synthesis on spectrally encoded beads for multiplexed protein/peptide bioassays

NASA Astrophysics Data System (ADS)

Nguyen, Huy Q.; Brower, Kara; Harink, Björn; Baxter, Brian; Thorn, Kurt S.; Fordyce, Polly M.

2017-02-01

Protein-peptide interactions are essential for cellular responses. Despite their importance, these interactions remain largely uncharacterized due to experimental challenges associated with their measurement. Current techniques (e.g. surface plasmon resonance, fluorescence polarization, and isothermal calorimetry) either require large amounts of purified material or direct fluorescent labeling, making high-throughput measurements laborious and expensive. In this report, we present a new technology for measuring antibody-peptide interactions in vitro that leverages spectrally encoded beads for biological multiplexing. Specific peptide sequences are synthesized directly on encoded beads with a 1:1 relationship between peptide sequence and embedded code, thereby making it possible to track many peptide sequences throughout the course of an experiment within a single small volume. We demonstrate the potential of these bead-bound peptide libraries by: (1) creating a set of 46 peptides composed of 3 commonly used epitope tags (myc, FLAG, and HA) and single amino-acid scanning mutants; (2) incubating with a mixture of fluorescently-labeled antimyc, anti-FLAG, and anti-HA antibodies; and (3) imaging these bead-bound libraries to simultaneously identify the embedded spectral code (and thus the sequence of the associated peptide) and quantify the amount of each antibody bound. To our knowledge, these data demonstrate the first customized peptide library synthesized directly on spectrally encoded beads. While the implementation of the technology provided here is a high-affinity antibody/protein interaction with a small code space, we believe this platform can be broadly applicable to any range of peptide screening applications, with the capability to multiplex into libraries of hundreds to thousands of peptides in a single assay.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

PubMed Central

Wan, Yuantai; Ma, Chengbang; Zhou, Mei; Xi, Xinping; Li, Lei; Wu, Di; Wang, Lei; Lin, Chen; Lopez, Juan Chavez; Chen, Tianbao; Shaw, Chris

2015-01-01

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities. PMID:26633506

Phylloseptin-PBa--A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity.

PubMed

Wan, Yuantai; Ma, Chengbang; Zhou, Mei; Xi, Xinping; Li, Lei; Wu, Di; Wang, Lei; Lin, Chen; Lopez, Juan Chavez; Chen, Tianbao; Shaw, Chris

2015-12-01

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.

PubMed

Tramontano, A; Bianchi, E; Venturini, S; Martin, F; Pessi, A; Sollazzo, M

1994-03-01

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.

Targeting mammalian organelles with internalizing phage (iPhage) libraries

PubMed Central

Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2015-01-01

Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

Determinants of BH3 Binding Specificity for Mcl-1 versus Bcl-x[subscript L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott

2010-06-25

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Prosurvival members of the family interact with proapoptotic BH3 (Bcl-2-homology-3)-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the {alpha}-helical BH3 region of the proapoptotic proteins to a conserved hydrophobic groove on the prosurvival proteins. Native BH3-only proteins exhibit selectivity in binding prosurvival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the designmore » of new classes of selective inhibitors to serve as reagents or therapeutics. In this work, we used two complementary techniques - yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis - to elucidate specificity determinants for binding to Bcl-x{sub L} versus Mcl-1, two prominent prosurvival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-x{sub L} selectively or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1-selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-x{sub L}, Bcl-2, Bcl-w, and Bfl-1, whereas Bcl-x{sub L}-selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 versus Bcl-x{sub L} binders.« less

Determinants of BH3 binding specificity for Mcl-1 vs. Bcl-xL

PubMed Central

Dutta, Sanjib; Gullá, Stefano; Chen, T. Scott; Fire, Emiko; Grant, Robert A.; Keating, Amy E.

2010-01-01

Interactions among Bcl-2 family proteins are important for regulating apoptosis. Pro-survival members of the family interact with pro-apoptotic BH3-only members, inhibiting execution of cell death through the mitochondrial pathway. Structurally, this interaction is mediated by binding of the alpha-helical BH3 region of the pro-apoptotic proteins to a conserved hydrophobic groove on the pro-survival proteins. Native BH3-only proteins exhibit selectivity in binding pro-survival members, as do small molecules that block these interactions. Understanding the sequence and structural basis of interaction specificity in this family is important, as it may allow the prediction of new Bcl-2 family associations and/or the design of new classes of selective inhibitors to serve as reagents or therapeutics. In this work we used two complementary techniques, yeast surface display screening from combinatorial peptide libraries and SPOT peptide array analysis, to elucidate specificity determinants for binding to Bcl-xL vs. Mcl-1, two prominent pro-survival proteins. We screened a randomized library and identified BH3 peptides that bound to either Mcl-1 or Bcl-xL selectively, or to both with high affinity. The peptides competed with native ligands for binding into the conserved hydrophobic groove, as illustrated in detail by a crystal structure of a specific peptide bound to Mcl-1. Mcl-1 selective peptides from the screen were highly specific for binding Mcl-1 in preference to Bcl-xL, Bcl-2, Bcl-w and Bfl-1, whereas Bcl-xL selective peptides showed some cross-interaction with related proteins Bcl-2 and Bcl-w. Mutational analyses using SPOT arrays revealed the effects of 170 point mutations made in the background of a peptide derived from the BH3 region of Bim, and a simple predictive model constructed using these data explained much of the specificity observed in our Mcl-1 vs. Bcl-xL binders. PMID:20363230

SABinder: A Web Service for Predicting Streptavidin-Binding Peptides.

PubMed

He, Bifang; Kang, Juanjuan; Ru, Beibei; Ding, Hui; Zhou, Peng; Huang, Jian

2016-01-01

Streptavidin is sometimes used as the intended target to screen phage-displayed combinatorial peptide libraries for streptavidin-binding peptides (SBPs). More often in the biopanning system, however, streptavidin is just a commonly used anchoring molecule that can efficiently capture the biotinylated target. In this case, SBPs creeping into the biopanning results are not desired binders but target-unrelated peptides (TUP). Taking them as intended binders may mislead subsequent studies. Therefore, it is important to find if a peptide is likely to be an SBP when streptavidin is either the intended target or just the anchoring molecule. In this paper, we describe an SVM-based ensemble predictor called SABinder. It is the first predictor for SBP. The model was built with the feature of optimized dipeptide composition. It was observed that 89.20% (MCC = 0.78; AUC = 0.93; permutation test, p < 0.001) of peptides were correctly classified. As a web server, SABinder is freely accessible. The tool provides a highly efficient way to exclude potential SBP when they are TUP or to facilitate identification of possibly new SBP when they are the desired binders. In either case, it will be helpful and can benefit related scientific community.

Monitoring multiple myeloma by idiotype-specific peptide binders of tumor-derived exosomes.

PubMed

Iaccino, Enrico; Mimmi, Selena; Dattilo, Vincenzo; Marino, Fabiola; Candeloro, Patrizio; Di Loria, Antonio; Marimpietri, Danilo; Pisano, Antonio; Albano, Francesco; Vecchio, Eleonora; Ceglia, Simona; Golino, Gaetanina; Lupia, Antonio; Fiume, Giuseppe; Quinto, Ileana; Scala, Giuseppe

2017-10-13

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression.

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro.

PubMed

Tonelli, R R; Colli, W; Alves, M J M

2012-01-01

Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, and Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques. The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development, and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment), were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction. In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic organisms will be discussed. Using these techniques, recent results on the interaction of Trypanosoma cruzi with the host will be highlighted focusing on members of the 85 kDa protein family, a subset of the gp85/TS superfamily.

Oligopeptide M13 Phage Display in Pathogen Research

PubMed Central

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-01-01

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline. PMID:24136040

Oligopeptide m13 phage display in pathogen research.

PubMed

Kügler, Jonas; Zantow, Jonas; Meyer, Torsten; Hust, Michael

2013-10-16

Phage display has become an established, widely used method for selection of peptides, antibodies or alternative scaffolds. The use of phage display for the selection of antigens from genomic or cDNA libraries of pathogens which is an alternative to the classical way of identifying immunogenic proteins is not well-known. In recent years several new applications for oligopeptide phage display in disease related fields have been developed which has led to the identification of various new antigens. These novel identified immunogenic proteins provide new insights into host pathogen interactions and can be used for the development of new diagnostic tests and vaccines. In this review we focus on the M13 oligopeptide phage display system for pathogen research but will also give examples for lambda phage display and for applications in other disease related fields. In addition, a detailed technical work flow for the identification of immunogenic oligopeptides using the pHORF system is given. The described identification of immunogenic proteins of pathogens using oligopeptide phage display can be linked to antibody phage display resulting in a vaccine pipeline.

Improving short antimicrobial peptides despite elusive rules for activity.

PubMed

Mikut, Ralf; Ruden, Serge; Reischl, Markus; Breitling, Frank; Volkmer, Rudolf; Hilpert, Kai

2016-05-01

Antimicrobial peptides (AMPs) can effectively kill a broad range of life threatening multidrug-resistant bacteria, a serious threat to public health worldwide. However, despite great hopes novel drugs based on AMPs are still rare. To accelerate drug development we studied different approaches to improve the antibacterial activity of short antimicrobial peptides. Short antimicrobial peptides seem to be ideal drug candidates since they can be synthesized quickly and easily, modified and optimized. In addition, manufacturing a short peptide drug will be more cost efficient than long and structured ones. In contrast to longer and structured peptides short AMPs seem hard to design and predict. Here, we designed, synthesized and screened five different peptide libraries, each consisting of 600 9-mer peptides, against Pseudomonas aeruginosa. Each library is presenting a different approach to investigate effectiveness of an optimization strategy. The data for the 3000 peptides were analyzed using models based on fuzzy logic bioinformatics and plausible descriptors. The rate of active or superior active peptides was improved from 31.0% in a semi-random library from a previous study to 97.8% in the best new designed library. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert. Copyright © 2015 Elsevier B.V. All rights reserved.

Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

PubMed

Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

2015-12-01

Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

Functionalization of semiconductors for biosensing applications

NASA Astrophysics Data System (ADS)

Estephan, E.; Larroque, C.; Martineau, P.; Cloitre, T.; Gergely, Cs.

2007-05-01

Functionalization of semiconductors (SC) has been widely used for various electronic, photonic and biomedical applications. In this paper, we report on selective functionalization achieved by peptides that reveal specific recognition of the SC surfaces. A M13 bacteriophage library was used to screen 10 10 different 12-mer peptide on various SC substrates to successfully isolate after 3 cycles one specific peptide for the majority of semiconductors. Our results conclude that GaAs(100) and GaN(0001) retain the same sequence of 12-mer peptide, suggesting that the specificity does not depend on the crystallographic structure but it depends on the chemical composition and the electronegativity of the surface, thus on the orientation of the material. We also note the presence of at least one proline (Pro) amino acid in each peptide, and the presence of the histidine (His) in the specific peptides for the II-VI class SC. Pro imprints a constraint to the peptide to facilitate adhesion to the surface, whereas the basic side chain His is known for its affinity towards some of the elements of class II SC. Finally, fluorescence microscopy has been employed to demonstrate the preferential attachment of the peptide to their specific SC surface in close proximity to a surface of different chemical and structural composition. The use of selected peptides expressed by phage display can be extended to encompass a variety of nanostructured semiconductor based devices.

Phage Display Technology in Biomaterials Engineering: Progress and Opportunities for Applications in Regenerative Medicine.

PubMed

Martins, Ivone M; Reis, Rui L; Azevedo, Helena S

2016-11-18

The field of regenerative medicine has been gaining momentum steadily over the past few years. The emphasis in regenerative medicine is to use various in vitro and in vivo approaches that leverage the intrinsic healing mechanisms of the body to treat patients with disabling injuries and chronic diseases such as diabetes, osteoarthritis, and degenerative disorders of the cardiovascular and central nervous system. Phage display has been successfully employed to identify peptide ligands for a wide variety of targets, ranging from relatively small molecules (enzymes, cell receptors) to inorganic, organic, and biological (tissues) materials. Over the past two decades, phage display technology has advanced tremendously and has become a powerful tool in the most varied fields of research, including biotechnology, materials science, cell biology, pharmacology, and diagnostics. The growing interest in and success of phage display libraries is largely due to its incredible versatility and practical use. This review discusses the potential of phage display technology in biomaterials engineering for applications in regenerative medicine.

Computationally assisted screening and design of cell-interactive peptides by a cell-based assay using peptide arrays and a fuzzy neural network algorithm.

PubMed

Kaga, Chiaki; Okochi, Mina; Tomita, Yasuyuki; Kato, Ryuji; Honda, Hiroyuki

2008-03-01

We developed a method of effective peptide screening that combines experiments and computational analysis. The method is based on the concept that screening efficiency can be enhanced from even limited data by use of a model derived from computational analysis that serves as a guide to screening and combining the model with subsequent repeated experiments. Here we focus on cell-adhesion peptides as a model application of this peptide-screening strategy. Cell-adhesion peptides were screened by use of a cell-based assay of a peptide array. Starting with the screening data obtained from a limited, random 5-mer library (643 sequences), a rule regarding structural characteristics of cell-adhesion peptides was extracted by fuzzy neural network (FNN) analysis. According to this rule, peptides with unfavored residues in certain positions that led to inefficient binding were eliminated from the random sequences. In the restricted, second random library (273 sequences), the yield of cell-adhesion peptides having an adhesion rate more than 1.5-fold to that of the basal array support was significantly high (31%) compared with the unrestricted random library (20%). In the restricted third library (50 sequences), the yield of cell-adhesion peptides increased to 84%. We conclude that a repeated cycle of experiments screening limited numbers of peptides can be assisted by the rule-extracting feature of FNN.

Biomolecule mediating synthesis of inorganic nanoparticles and their applications

NASA Astrophysics Data System (ADS)

Wei, Zengyan

Project 1. The conventional phage display technique focuses on screening peptide sequences that can bind on target substrates, however the selected peptides are not necessary to nucleate and mediate the growth of the target inorganic crystals, and in many cases they only show moderate affinity to the targets. Here we report a novel phage display approach that can directly screen peptides catalytically growing inorganic nanoparticles in aqueous solution at room temperature. In this study, the phage library is incubated with zinc precursor at room temperature. Among random peptide sequences displayed on phages, those phages that can grow zinc oxide (ZnO) nanoparticles are selected with centrifugation. After several rounds of selection, the peptide sequences displayed on the phage viruses are analyzed by DNA sequencing. Our screening protocol provide a simple and convenient route for the discovery of catalytic peptides that can grow inorganic nanoparticles at room temperature. This novel screening protocol can extend the method on finding a wide range of new catalysts. Project 2. Genetically engineered collagen peptides are assembled into freestanding films when quantum dots (QDs) are co-assembled as joints between collagen domains. These peptide-based films show excellent mechanical properties with Young's modulus of 20 GPa, much larger than most of the multi-composite polymer films and previously reported freestanding nanoparticle-assembled sheets, and it is even close to that reported for the bone tissue in nature. These films show little permanent deformation under small indentation while the mechanical hysteresis becomes remarkable when the load approaches near and beyond the rupture point, which is also characteristic of the bone tissue. Project 3. The shape-controlled synthesis of nanoparticles have been established in single-phase solutions by controlling growth directions of crystalline facets on seed nanocrystals kinetically; however, it is difficult to rationally predict and design nanoparticle shapes. Here we introduce a methodology to fabricate nanoparticles in smaller sizes by evolving shapes thermodynamically. This strategy enables a more rational approach to fabricate shaped nanoparticles by etching specific positions of atoms on facets of seed nanocrystals in reverse micelle reactors where the surface energy gradient induces desorption of atoms on specific locations on the seed surfaces. From seeds of 12 nm palladium nanocubes, the shape is evolved to concave nanocubes and finally hollow nanocages in the size 10 nm by etching the center of {200} facets. The high surface area-to-volume ratio and the exposure of a large number of palladium atoms on ledge and kink sites of hollow nanocages are advantageous to enhance catalytic activity and recyclability.

Selection of antitumor displayed peptides for the specific delivery of the anticancer drug lactaptin

PubMed Central

Nemudraya, Anna Andreevna; Kuligina, Elena Vladimirovna; Ilyichev, Alexandr Alexeevich; Fomin, Alexandr Sergeevich; Stepanov, Grigory Alexandrovich; Savelyeva, Anna Valentinovna; Koval, Olga Alexandrovna; Richter, Vladimir Alexandrovich

2016-01-01

It has been previously demonstrated that lactaptin, the proteolytic fragment of human milk protein κ-casein, induces the death of various cultured cancer cells. The recombinant analog of lactaptin, RL2, effectively induces the apoptosis of mouse hepatocarcinoma-1 (HA-1) tumor cells in vitro and suppress the growth of HA-1 tumors and metastases in vivo. The antitumor drug Lactaptin developed on the basis of RL2 has been successful in preclinical trials. Lactaptin shows its efficiency in relation to mouse and human cancer cells and tumors. However, Lactaptin, as with the majority of protein-based therapeutic drugs, is distributed evenly throughout the organism, which reduces its antitumor efficacy. To develop the targeted delivery of lactaptin, the present study selected tumor-specific peptides by screening a phage display peptide library in vivo on A/Sn strain mice with subcutaneously transplanted HA-1 cells. Two genetic constructs were made for the production of recombinant fusion proteins composed of RL2 and the selected tumor-targeting peptide. In vitro experiments involving HA-1, MDA-MB-231 and MCF-7 cells cultures demonstrated that the fusion proteins induce apoptotic death in mouse and human tumor cells, as with RL2. The in vivo experiments involving the mouse HA-1 tumor model demonstrated that the tumor fluorescence intensity of the Cy5-fusion protein conjugates is higher than that of RL2-Cy5. As conjugation of the tumor-specific peptides to RL2 provided retention of RL2 in the tumor tissues, fusion proteins composed of lactaptin and peptides specific for human tumors are deemed promising to improve the antitumor efficiency of lactaptin. PMID:28105163

A new strategy for the preparation of peptide-targeted technetium and rhenium radiopharmaceuticals. The automated solid-phase synthesis, characterization, labeling, and screening of a peptide-ligand library targeted at the formyl peptide receptor.

PubMed

Stephenson, Karin A; Banerjee, Sangeeta Ray; Sogbein, Oyebola O; Levadala, Murali K; McFarlane, Nicole; Boreham, Douglas R; Maresca, Kevin P; Babich, John W; Zubieta, Jon; Valliant, John F

2005-01-01

A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-alpha-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the K(d) values were found to be in the low micromolar range (0.5-3.0 microM). Compound 5j was subsequently labeled with (99m)Tc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization.

PubMed

Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P; Mirokhin, Yuri A; Tchekhovskoi, Dmitrii V; Bukhari, Tallat H; Stein, Stephen E

2018-04-01

We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins.

The NISTmAb tryptic peptide spectral library for monoclonal antibody characterization

PubMed Central

Dong, Qian; Liang, Yuxue; Yan, Xinjian; Markey, Sanford P.; Mirokhin, Yuri A.; Tchekhovskoi, Dmitrii V.; Bukhari, Tallat H.; Stein, Stephen E.

2018-01-01

ABSTRACT We describe the creation of a mass spectral library composed of all identifiable spectra derived from the tryptic digest of the NISTmAb IgG1κ. The library is a unique reference spectral collection developed from over six million peptide-spectrum matches acquired by liquid chromatography-mass spectrometry (LC-MS) over a wide range of collision energy. Conventional one-dimensional (1D) LC-MS was used for various digestion conditions and 20- and 24-fraction two-dimensional (2D) LC-MS studies permitted in-depth analyses of single digests. Computer methods were developed for automated analysis of LC-MS isotopic clusters to determine the attributes for all ions detected in the 1D and 2D studies. The library contains a selection of over 12,600 high-quality tandem spectra of more than 3,300 peptide ions identified and validated by accurate mass, differential elution pattern, and expected peptide classes in peptide map experiments. These include a variety of biologically modified peptide spectra involving glycosylated, oxidized, deamidated, glycated, and N/C-terminal modified peptides, as well as artifacts. A complete glycation profile was obtained for the NISTmAb with spectra for 58% and 100% of all possible glycation sites in the heavy and light chains, respectively. The site-specific quantification of methionine oxidation in the protein is described. The utility of this reference library is demonstrated by the analysis of a commercial monoclonal antibody (adalimumab, Humira®), where 691 peptide ion spectra are identifiable in the constant regions, accounting for 60% coverage for both heavy and light chains. The NIST reference library platform may be used as a tool for facile identification of the primary sequence and post-translational modifications, as well as the recognition of LC-MS method-induced artifacts for human and recombinant IgG antibodies. Its development also provides a general method for creating comprehensive peptide libraries of individual proteins. PMID:29425077

Ligand-targeted theranostic nanomedicines against cancer

DOE PAGES

Yao, Virginia J.; D'Angelo, Sara; Butler, Kimberly S.; ...

2016-01-06

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20 years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentiallymore » overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. As a result, the modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.« less

Ligand-targeted theranostic nanomedicines against cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Virginia J.; D'Angelo, Sara; Butler, Kimberly S.

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20 years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentiallymore » overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. As a result, the modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.« less

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

PubMed

Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

2016-01-01

Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

Screening of Peptide Libraries Against Protective Antigen of Bacillus Anthracis in a Disposable Microfluidic Cartridge

DTIC Science & Technology

2011-11-28

New Reprint Screening of Peptide Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge W911NF-09-D-0001...against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Report Title ABSTRACT See attached. Screening of Peptide...Libraries against Protective Antigen of Bacillus anthracis in a Disposable Microfluidic Cartridge Joshua M. Kogot1, Yanting Zhang2, Stephen J. Moore3

Building high-quality assay libraries for targeted analysis of SWATH MS data.

PubMed

Schubert, Olga T; Gillet, Ludovic C; Collins, Ben C; Navarro, Pedro; Rosenberger, George; Wolski, Witold E; Lam, Henry; Amodei, Dario; Mallick, Parag; MacLean, Brendan; Aebersold, Ruedi

2015-03-01

Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS. We describe each step of the process, including data acquisition by discovery proteomics, assertion of peptide-spectrum matches (PSMs), generation of consensus spectra and compilation of MS coordinates that uniquely define each targeted peptide. Crucial steps such as false discovery rate (FDR) control, retention time normalization and handling of post-translationally modified peptides are detailed. Finally, we show how to use the library to extract SWATH data with the open-source software Skyline. The protocol takes 2-3 d to complete, depending on the extent of the library and the computational resources available.

PECAN: library-free peptide detection for data-independent acquisition tandem mass spectrometry data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ting, Ying S.; Egertson, Jarrett D.; Bollinger, James G.

Data-independent acquisition (DIA) is an emerging mass spectrometry (MS)-based technique for unbiased and reproducible measurement of protein mixtures. DIA tandem mass spectrometry spectra are often highly multiplexed, containing product ions from multiple cofragmenting precursors. Detecting peptides directly from DIA data is therefore challenging; most DIA data analyses require spectral libraries. Here we present PECECAN (http://pecan.maccosslab.org), a library-free, peptide-centric tool that robustly and accurately detects peptides directly from DIA data. PECECAN reports evidence of detection based on product ion scoring, which enables detection of low-abundance analytes with poor precursor ion signal. We demonstrate the chromatographic peak picking accuracy and peptide detectionmore » capability of PECECAN, and we further validate its detection with data-dependent acquisition and targeted analyses. Lastly, we used PECECAN to build a plasma proteome library from DIA data and to query known sequence variants.« less

Biomaterials functionalization using a novel peptide that selectively binds to a conducting polymer

NASA Astrophysics Data System (ADS)

Sanghvi, Archit B.; Miller, Kiley P.-H.; Belcher, Angela M.; Schmidt, Christine E.

2005-06-01

The goal in biomaterial surface modification is to retain a material's bulk properties while modifying only its surface to possess desired recognition and specificity. Here we develop a unique strategy for surface functionalization of an electrically conductive polymer, chlorine-doped polypyrrole (PPyCl), which has been widely researched for various electronic and biomedical applications. An M13 bacteriophage library was used to screen 109 different 12-mer peptide inserts against PPyCl. A binding phage (ϕT59) was isolated, and its binding stability and specificity to PPyCl was assessed using fluorescence microscopy and titer count analysis. The relative binding strength and mechanism of the corresponding 12-mer peptide and its variants was studied using atomic force microscopy and fluorescamine assays. Further, the T59 peptide was joined to a cell adhesive sequence and used to promote cell attachment on PPyCl. This strategy can be extended to immobilize a variety of molecules to PPyCl for numerous applications. In addition, phage display can be applied to other polymers to develop bioactive materials without altering their bulk properties.

Spectrum-based method to generate good decoy libraries for spectral library searching in peptide identifications.

PubMed

Cheng, Chia-Ying; Tsai, Chia-Feng; Chen, Yu-Ju; Sung, Ting-Yi; Hsu, Wen-Lian

2013-05-03

As spectral library searching has received increasing attention for peptide identification, constructing good decoy spectra from the target spectra is the key to correctly estimating the false discovery rate in searching against the concatenated target-decoy spectral library. Several methods have been proposed to construct decoy spectral libraries. Most of them construct decoy peptide sequences and then generate theoretical spectra accordingly. In this paper, we propose a method, called precursor-swap, which directly constructs decoy spectral libraries directly at the "spectrum level" without generating decoy peptide sequences by swapping the precursors of two spectra selected according to a very simple rule. Our spectrum-based method does not require additional efforts to deal with ion types (e.g., a, b or c ions), fragment mechanism (e.g., CID, or ETD), or unannotated peaks, but preserves many spectral properties. The precursor-swap method is evaluated on different spectral libraries and the results of obtained decoy ratios show that it is comparable to other methods. Notably, it is efficient in time and memory usage for constructing decoy libraries. A software tool called Precursor-Swap-Decoy-Generation (PSDG) is publicly available for download at http://ms.iis.sinica.edu.tw/PSDG/.

Affinity reagent technology development and application to rapid immunochromatographic pathogen detection

NASA Astrophysics Data System (ADS)

Sooter, Letha J.; Stratis-Cullum, Dimitra N.; Zhang, Yanting; Daugherty, Patrick S.; Soh, H. Tom; Pellegrino, Paul; Stagliano, Nancy

2007-09-01

Immunochromatography is a rapid, reliable, and cost effective method of detecting biowarfare agents. The format is similar to that of an over-the-counter pregnancy test. A sample is applied to one end of a cassette and then a control line, and possibly a sample line, are visualized at the other end of the cassette. The test is based upon a sandwich assay. For the control, a line of Protein A is immobilized on the membrane. Gold nanoparticle bound IgG flows through the membrane and binds the Protein A, creating a visible line on the membrane. For the sample, one epitope is immobilized on the membrane and another epitope is attached to gold nanoparticles. The sample binds gold bound epitope, travels through the membrane, and binds membrane bound epitope. The two epitopes are not cross-reactive, therefore a sample line is only visible if the sample is present. In order to efficiently screen for binders to a sample target, a novel, Continuous Magnetic Activated Cell Sorter (CMACS) has been developed on a disposable, microfluidic platform. The CMACS chip quickly sorts E. coli peptide libraries for target binders with high affinity. Peptide libraries, are composed of approximately ten million bacteria, each displaying a different peptide on their surface. The target of interest is conjugated to a micrometer sized magnetic particle. After the library and the target are incubated together to allow binding, the mixture is applied to the CMACS chip. In the presence of patterned nickel and an external magnet, separation occurs of the bead-bound bacteria from the bulk material. The bead fraction is added to bacterial growth media where any attached E. coli grow and divide. These cells are cloned, sequenced, and the peptides are assayed for target binding affinity. As a proof-of-principle, assays were developed for human C-reactive protein. More defense relevant targets are currently being pursued.

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

PubMed Central

Staquicini, Fernanda I.; Ozawa, Michael G.; Moya, Catherine A.; Driessen, Wouter H.P.; Barbu, E. Magda; Nishimori, Hiroyuki; Soghomonyan, Suren; Flores, Leo G.; Liang, Xiaowen; Paolillo, Vincenzo; Alauddin, Mian M.; Basilion, James P.; Furnari, Frank B.; Bogler, Oliver; Lang, Frederick F.; Aldape, Kenneth D.; Fuller, Gregory N.; Höök, Magnus; Gelovani, Juri G.; Sidman, Richard L.; Cavenee, Webster K.; Pasqualini, Renata; Arap, Wadih

2010-01-01

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors. PMID:21183793

Phage display as a promising approach for vaccine development.

PubMed

Aghebati-Maleki, Leili; Bakhshinejad, Babak; Baradaran, Behzad; Motallebnezhad, Morteza; Aghebati-Maleki, Ali; Nickho, Hamid; Yousefi, Mehdi; Majidi, Jafar

2016-09-29

Bacteriophages are specific antagonists to bacterial hosts. These viral entities have attracted growing interest as optimal vaccine delivery vehicles. Phages are well-matched for vaccine design due to being highly stable under harsh environmental conditions, simple and inexpensive large scale production, and potent adjuvant capacities. Phage vaccines have efficient immunostimulatory effects and present a high safety profile because these viruses have made a constant relationship with the mammalian body during a long-standing evolutionary period. The birth of phage display technology has been a turning point in the development of phage-based vaccines. Phage display vaccines are made by expressing multiple copies of an antigen on the surface of immunogenic phage particles, thereby eliciting a powerful and effective immune response. Also, the ability to produce combinatorial peptide libraries with a highly diverse pool of randomized ligands has transformed phage display into a straightforward, versatile and high throughput screening methodology for the identification of potential vaccine candidates against different diseases in particular microbial infections. These libraries can be conveniently screened through an affinity selection-based strategy called biopanning against a wide variety of targets for the selection of mimotopes with high antigenicity and immunogenicity. Also, they can be panned against the antiserum of convalescent individuals to recognize novel peptidomimetics of pathogen-related epitopes. Phage display has represented enormous promise for finding new strategies of vaccine discovery and production and current breakthroughs promise a brilliant future for the development of different phage-based vaccine platforms.

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals.

PubMed

Cho, Whirang; Fowler, Jeffrey D; Furst, Eric M

2012-04-10

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity. © 2012 American Chemical Society

Asymmetric triplex metallohelices with high and selective activity against cancer cells

NASA Astrophysics Data System (ADS)

Faulkner, Alan D.; Kaner, Rebecca A.; Abdallah, Qasem M. A.; Clarkson, Guy; Fox, David J.; Gurnani, Pratik; Howson, Suzanne E.; Phillips, Roger M.; Roper, David I.; Simpson, Daniel H.; Scott, Peter

2014-09-01

Small cationic amphiphilic α-helical peptides are emerging as agents for the treatment of cancer and infection, but they are costly and display unfavourable pharmacokinetics. Helical coordination complexes may offer a three-dimensional scaffold for the synthesis of mimetic architectures. However, the high symmetry and modest functionality of current systems offer little scope to tailor the structure to interact with specific biomolecular targets, or to create libraries for phenotypic screens. Here, we report the highly stereoselective asymmetric self-assembly of very stable, functionalized metallohelices. Their anti-parallel head-to-head-to-tail ‘triplex’ strand arrangement creates an amphipathic functional topology akin to that of the active sub-units of, for example, host-defence peptides and p53. The metallohelices display high, structure-dependent toxicity to the human colon carcinoma cell-line HCT116 p53++, causing dramatic changes in the cell cycle without DNA damage. They have lower toxicity to human breast adenocarcinoma cells (MDA-MB-468) and, most remarkably, they show no significant toxicity to the bacteria methicillin-resistant Staphylococcus aureus and Escherichia coli.

Invert biopanning: A novel method for efficient and rapid isolation of scFvs by phage display technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Tanomand, Asghar; Akbari, Bahman

2016-11-01

Phage display is a prominent screening technique for development of novel high affinity antibodies against almost any antigen. However, removing false positive clones in screening process remains a challenge. The aim of this study was to develop an efficient and rapid method for isolation of high affinity scFvs by removing NSBs without losing rare specific clones. Therefore, a novel two rounds strategy called invert biopanning was developed for isolating high affinity scFvs against EGFRvIII antigen from human scFv library. The efficiency of invert biopanning method (procedure III) was analyzed by comparing with results of conventional biopanning methods (procedures I and II). According to the results of polyclonal ELISA, the second round of procedure III displayed highest binding affinity against EGFRvIII peptide accompanied by lowest NSB comparing to other two procedures. Several positive clones were identified among output phages of procedure III by monoclonal phage ELISA which displayed high affinity to EGFRvIII antigen. In conclusion, results of our study indicate that invert biopanning is an efficient method for avoiding NSBs and conservation of rare specific clones during screening of a scFv phage library. Novel anti EGFRvIII scFv isolated could be a promising candidate for potential use in treatment of EGFRvIII expressing cancers. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry.

PubMed

Gurevich-Messina, Juan M; Giudicessi, Silvana L; Martínez-Ceron, María C; Acosta, Gerardo; Erra-Balsells, Rosa; Cascone, Osvaldo; Albericio, Fernando; Camperi, Silvia A

2015-01-01

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of 'one-bead-one-peptide' combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4-hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc-Asp[2-phenylisopropyl (OPp)]-OH to Ala-Gly-oxymethylbenzamide-ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N-terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N-Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one-bead-one-cyclic depsipeptide libraries that can be easily open for its sequencing by matrix-assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Viral assembly of oriented quantum dot nanowires

NASA Astrophysics Data System (ADS)

Mao, Chuanbin; Flynn, Christine E.; Hayhurst, Andrew; Sweeney, Rozamond; Qi, Jifa; Georgiou, George; Iverson, Brent; Belcher, Angela M.

2003-06-01

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Viral assembly of oriented quantum dot nanowires.

PubMed

Mao, Chuanbin; Flynn, Christine E; Hayhurst, Andrew; Sweeney, Rozamond; Qi, Jifa; Georgiou, George; Iverson, Brent; Belcher, Angela M

2003-06-10

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Construction of a high efficiency copper adsorption bacterial system via peptide display and its application on copper dye polluted wastewater.

PubMed

Maruthamuthu, Murali Kannan; Nadarajan, Saravanan Prabhu; Ganesh, Irisappan; Ravikumar, Sambandam; Yun, Hyungdon; Yoo, Ik-Keun; Hong, Soon Ho

2015-11-01

For the construction of an efficient copper waste treatment system, a cell surface display strategy was employed. The copper adsorption ability of recombinant bacterial strains displaying three different copper binding peptides were evaluated in LB Luria-Bertani medium (LB), artificial wastewater, and copper phthalocyanine containing textile dye industry wastewater samples. Structural characteristics of the three peptides were also analyzed by similarity-based structure modeling. The best binding peptide was chosen for the construction of a dimeric peptide display and the adsorption ability of the monomeric and dimeric peptide displayed strains were compared. The dimeric peptide displayed strain showed superior copper adsorption in all three tested conditions (LB, artificial wastewater, and textile dye industry wastewater). When the strains were exposed to copper phthalocyanine dye polluted wastewater, the dimeric peptide display [543.27 µmol/g DCW dry cell weight (DCW)] showed higher adsorption of copper when compared with the monomeric strains (243.53 µmol/g DCW).

Uncovering the design rules for peptide synthesis of metal nanoparticles.

PubMed

Tan, Yen Nee; Lee, Jim Yang; Wang, Daniel I C

2010-04-28

Peptides are multifunctional reagents (reducing and capping agents) that can be used for the synthesis of biocompatible metal nanoparticles under relatively mild conditions. However, the progress in peptide synthesis of metal nanoparticles has been slow due to the lack of peptide design rules. It is difficult to establish sequence-reactivity relationships from peptides isolated from biological sources (e.g., biomineralizing organisms) or selected by combinatorial display libraries because of their widely varying compositions and structures. The abundance of random and inactive amino acid sequences in the peptides also increases the difficulty in knowledge extraction. In this study, a "bottom-up" approach was used to formulate a set of rudimentary rules for the size- and shape-controlled peptide synthesis of gold nanoparticles from the properties of the 20 natural alpha-amino acids for AuCl(4)(-) reduction and binding to Au(0). It was discovered that the reduction capability of a peptide depends on the presence of certain reducing amino acid residues, whose activity may be regulated by neighboring residues with different Au(0) binding strengths. Another finding is the effect of peptide net charge on the nucleation and growth of the Au nanoparticles. On the basis of these understandings, several multifunctional peptides were designed to synthesize gold nanoparticles in different morphologies (nanospheres and nanoplates) and with sizes tunable by the strategic placement of selected amino acid residues in the peptide sequence. The methodology presented here and the findings are useful for establishing the scientific basis for the rational design of peptides for the synthesis of metal nanostructures.

Fas-associated Protein with Death Domain (FADD)-independent Recruitment of c-FLIPL to Death Receptor 5*

PubMed Central

Jin, Tai-Guang; Kurakin, Alexei; Benhaga, Nordine; Abe, Karon; Mohseni, Mehrdad; Sandra, Ferry; Song, Keli; Kay, Brian K.; Khosravi-Far, Roya

2010-01-01

Here we show a novel mechanism by which FLICE-like inhibitory protein (c-FLIP) regulates apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and one of its receptors, DR5. c-FLIP is a critical regulator of the TNF family of cytokine receptor signaling. c-FLIP has been postulated to prevent formation of the competent death-inducing signaling complex (DISC) in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. In order to identify regulators of TRAIL function, we used the intracellular death domain (DD) of DR5 as a target to screen a phage-displayed combinatorial peptide library. The DD of DR5 selected from the library a peptide that showed sequence similarity to a stretch of amino acids in the C terminus of c-FLIPL. The phage-displayed peptide selectively interacted with the DD of DR5 in in vitro binding assays. Similarly, full-length c-FLIP (c-FLIPL) and the C-terminal p12 domain of c-FLIP interacted with DR5 both in in vitro pull-down assays and in mammalian cells. This interaction was independent of TRAIL. To the contrary, TRAIL treatment released c-FLIPL from DR5, permitting the recruitment of FADD to the active DR5 signaling complex. By employing FADD-deficient Jurkat cells, we demonstrate that DR5 and c-FLIPL interact in a FADD-independent manner. Moreover, we show that a cellular membrane permeable version of the peptide corresponding to the DR5 binding domain of c-FLIP induces apoptosis in mammalian cells. Taken together, these findings indicate that c-FLIPL interacts with the DD of DR5, thus preventing death signaling by DR5 prior to the formation of an active DISC. Because TRAIL and DR5 are ubiquitously expressed, the interaction of c-FLIPL and DR5 indicates a mechanism by which tumor selective apoptosis can be achieved through protecting normal cells from undergoing death receptor-induced apoptosis. PMID:15485835

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

PubMed

Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

1995-01-01

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

PubMed Central

Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

2017-01-01

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.

PubMed

Kunys, Andrew R; Lian, Wenlong; Pei, Dehua

2012-12-01

One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

PubMed

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

2017-03-11

Amino-acid mutations of Gly 12 (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH 2 ) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K D and IC 50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH 2 ) that inhibited enzyme activity of K-Ras(G12D) with IC 50  = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro selection using a dual RNA library that allows primerless selection

PubMed Central

Jarosch, Florian; Buchner, Klaus; Klussmann, Sven

2006-01-01

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. PMID:16855281

Definition of agonists and design of antagonists for alloreactive T cell clones using synthetic peptide libraries.

PubMed

de Koster, H S; Vermeulen, C J; Hiemstra, H S; Amons, R; Drijfhout, J W; Koning, F

1999-04-01

Alloreactive T cells form an important barrier for organ transplantation. To reduce the risk of rejection patients are given immunosuppressive drugs, which increase the chance of infection and the incidence of malignancies. It has been shown that a large proportion of alloreactive T cells specifically recognize peptides present in the groove of the allogeneic MHC molecule. This implies that it might be possible to modulate the alloresponse by peptides with antagonistic properties, thus preventing rejection without the side effects of general immunosuppression. Peptide antagonists can be designed on the basis of the original agonist, yet for alloreactive T cells these agonists are usually unknown. In this study we have used a dedicated synthetic peptide library to identify agonists for HLA-DR3-specific alloreactive T cell clones. Based on these agonists, altered peptide ligands (APL) were designed. Three APL could antagonize an alloreactive T cell clone in its response against the library-derived agonist as well as in its response against the original allodeterminant, HLA-DR3. This demonstrates that peptide libraries can be used to design antagonists for alloreactive T cells without knowledge about the nature of the actual allostimulatory peptide. Since the most potent agonists are selected, this strategy permits detection of potent antagonists. The results, however, also suggest that the degree of peptide dependency of alloreactive T cell clones may dictate whether a peptide antagonist can be found for such clones. Whether peptide antagonists will be valuable in the development of donor-patient-specific immunosuppression may therefore depend on the specificity of the in vivo-generated alloreactive T cells.

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos

PubMed Central

David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick

2018-01-01

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998

Specific probe selection from landscape phage display library and its application in enzyme-linked immunosorbent assay of free prostate-specific antigen.

PubMed

Lang, Qiaolin; Wang, Fei; Yin, Long; Liu, Mingjun; Petrenko, Valery A; Liu, Aihua

2014-03-04

Probes against targets can be selected from the landscape phage library f8/8, displaying random octapeptides on the pVIII coat protein of the phage fd-tet and demonstrating many excellent features including multivalency, stability, and high structural homogeneity. Prostate-specific antigen (PSA) is usually determined by immunoassay, by which antibodies are frequently used as the specific probes. Herein we found that more advanced probes against free prostate-specific antigen (f-PSA) can be screened from the landscape phage library. Four phage monoclones were selected and identified by the specificity array. One phage clone displaying the fusion peptide ERNSVSPS showed good specificity and affinity to f-PSA and was used as a PSA capture probe in a sandwich enzyme-linked immunosorbent assay (ELISA) array. An anti-human PSA monoclonal antibody (anti-PSA mAb) was used to recognize the captured antigen, followed by horseradish peroxidase-conjugated antibody (HRP-IgG) and o-phenylenediamine, which were successively added to develop plate color. The ELISA conditions such as effect of blocking agent, coating buffer pH, phage concentration, antigen incubation time, and anti-PSA mAb dilution for phage ELISA were optimized. On the basis of the optimal phage ELISA conditions, the absorbance taken at 492 nm on a microplate reader was linear with f-PSA concentration within 0.825-165 ng/mL with a low limit of detection of 0.16 ng/mL. Thus, the landscape phage is an attractive biomolecular probe in bioanalysis.

Epitope mapping of epidermal growth factor receptor (EGFR) monoclonal antibody and induction of growth-inhibitory polyclonal antibodies by vaccination with EGFR mimotope.

PubMed

Navari, Mohsen; Zare, Mehrak; Javanmardi, Masoud; Asadi-Ghalehni, Majid; Modjtahedi, Helmout; Rasaee, Mohammad Javed

2014-10-01

One of the proposed approaches in cancer therapy is to induce and direct the patient's own immune system against cancer cells. In this study, we determined the epitope mapping of the rat anti-human epidermal growth factor receptor (EGFR) monoclonal antibody ICR-62 using a phage display of random peptide library and identified a 12 amino acids peptide, which was recognized as a mimotope. The peptide was synthesized and conjugated to bovine serum albumin (BSA) as carrier protein (P-BSA). We have shown that ICR-62 can react specifically with P-BSA as well as native EGFR. Two rabbits were immunized either by BSA or P-BSA and the rabbits IgGs were purified and examined for binding to the antigens, mimotope and the EGFR protein purified from the EGFR overexpressing A431 cell line. We showed that the rabbit IgG generated against the mimotope is capable of inhibiting the growth of A431 cells by 15%, but does not have any effect on the growth of EGFR-negative MDA-MB-453 cell line in vitro. Our results support the need for further investigations on the potential of vaccination with either mimotope of the EGFR or epitope displayed on the surface of phage particles for use in active immunotherapy of cancer.

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

PubMed Central

He, M; Taussig, M J

1997-01-01

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries. PMID:9396828

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

PubMed

He, M; Taussig, M J

1997-12-15

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.

Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

PubMed Central

2015-01-01

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme. PMID:24841702

PNA-encoded chemical libraries.

PubMed

Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

2015-06-01

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70. Copyright © 2015 Elsevier Ltd. All rights reserved.

T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones.

PubMed

Theaker, Sarah M; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J; Cole, David K; Peakman, Mark; Sewell, Andrew K; Dolton, Garry

2016-03-01

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8(+) or CD4(+) polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein-Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.

PubMed

Garbe, Daniel; Thiel, Ilka V; Mootz, Henning D

2010-10-01

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

A strategy for actualization of active targeting nanomedicine practically functioning in a living body.

PubMed

Lee, Kyoung Jin; Shin, Seol Hwa; Lee, Jae Hee; Ju, Eun Jin; Park, Yun-Yong; Hwang, Jung Jin; Suh, Young-Ah; Hong, Seung-Mo; Jang, Se Jin; Lee, Jung Shin; Song, Si Yeol; Jeong, Seong-Yun; Choi, Eun Kyung

2017-10-01

Designing nanocarriers with active targeting has been increasingly emphasized as for an ideal delivery mechanism of anti-cancer therapeutic agents, but the actualization has been constrained by lack of reliable strategy ultimately applicable. Here, we designed and verified a strategy to achieve active targeting nanomedicine that works in a living body, utilizing animal models bearing a patient's tumor tissue and subjected to the same treatments that would be used in the clinic. The concept for this strategy was that a novel peptide probe and its counterpart protein, which responded to a therapy, were identified, and then the inherent ability of the peptide to target the designated tumor protein was used for active targeting in vivo. An initial dose of ionizing radiation was locally delivered to the gastric cancer (GC) tumor of a patient-derived xenograft mouse model, and phage-displayed peptide library was intravenously injected. The peptides tightly bound to the tumor were recovered, and the counterpart protein was subsequently identified. Peptide-conjugated liposomal drug showed dramatically improved therapeutic efficacy and possibility of diagnostic imaging with radiation. These results strongly suggested the potential of our strategy to achieve in vivo functional active targeting and to be applied clinically for human cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment

PubMed Central

Dasari, Surendra; Chambers, Matthew C.; Martinez, Misti A.; Carpenter, Kristin L.; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo J.; Tabb, David L.

2012-01-01

Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines. PMID:22217208

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, S.-S.; IGE Therapeutics, Inc., Cellular and Cancer Immunology, 6370 Lusk Boulevard, F109, San Diego, CA 92121; Yang Yongmin

GFP-C{kappa} fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 {mu}g of the dual site aptameric GFP library exhibiting amore » diversity of 7.5 x 10{sup 11} was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.« less

Phage display and molecular imaging: expanding fields of vision in living subjects.

PubMed

Cochran, R; Cochran, Frank

2010-01-01

In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use of phage particles as imaging agents is also described. In the latter part of the review, a survey of phage-derived in vivo imaging agents is presented, and important recent examples are highlighted. Other imaging applications are also discussed, such as the development of peptide tags for site-specific protein labeling and the use of phage as delivery agents for reporter genes. The review concludes with a discussion of how phage display technology will continue to impact both basic science and clinical applications in the field of molecular imaging.

Catalytic applications of bio-inspired nanomaterials

NASA Astrophysics Data System (ADS)

Pacardo, Dennis Kien Balaong

The biomimetic synthesis of Pd nanoparticles was presented using the Pd4 peptide, TSNAVHPTLRHL, isolated from combinatorial phage display library. Using this approach, nearly monodisperse and spherical Pd nanoparticles were generated with an average diameter of 1.9 +/- 0.4 nm. The peptide-based nanocatalyst were employed in the Stille coupling reaction under energy-efficient and environmentally friendly reaction conditions of aqueous solvent, room temperature and very low catalyst loading. To this end, the Pd nanocatalyst generated high turnover frequency (TOF) value and quantitative yields using ≥ 0.005 mol% Pd as well as catalytic activities with different aryl halides containing electron-withdrawing and electron-donating groups. The Pd4-capped Pd nanoparticles followed the atom-leaching mechanism and were found to be selective with respect to substrate identity. On the other hand, the naturally-occurring R5 peptide (SSKKSGSYSGSKGSKRRIL) was employed in the synthesis of biotemplated Pd nanomaterials which showed morphological changes as a function of Pd:peptide ratio. TOF analysis for hydrogenation of olefinic alcohols showed similar catalytic activity regardless of nanomorphology. Determination of catalytic properties of these bio-inspired nanomaterials are important as they serve as model system for alternative green catalyst with applications in industrially important transformations.

K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya

Amino-acid mutations of Gly{sup 12} (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH{sub 2}) as a consensus sequence. KRpep-2 showedmore » more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. K{sub D} and IC{sub 50} values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH{sub 2}) that inhibited enzyme activity of K-Ras(G12D) with IC{sub 50} = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs. - Highlights: • The first K-Ras(G12D)-selective inhibitory peptides were generated. • These peptides showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D) in compared to wild type K-Ras. • The peptide KRpep-2d suppressed downstream signal of K-Ras(G12D) and cell proliferations of cancer cell line A427.« less

Ligand-targeted theranostic nanomedicines against cancer.

PubMed

Yao, Virginia J; D'Angelo, Sara; Butler, Kimberly S; Theron, Christophe; Smith, Tracey L; Marchiò, Serena; Gelovani, Juri G; Sidman, Richard L; Dobroff, Andrey S; Brinker, C Jeffrey; Bradbury, Andrew R M; Arap, Wadih; Pasqualini, Renata

2016-10-28

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

PubMed

Gardères, Johan; Domart-Coulon, Isabelle; Marie, Arul; Hamer, Bojan; Batel, Renato; Müller, Werner E G; Bourguet-Kondracki, Marie-Lise

2016-10-01

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

Selection of phage-displayed peptides for the detection of imidacloprid in water and soil.

PubMed

Liu, Zhiping; Liu, Jianfeng; Wang, Kai; Li, Wenhui; Shelver, Weilin L; Li, Qing X; Li, Ji; Xu, Ting

2015-09-15

Imidacloprid is the most widely used neonicotinoid insecticide in the world and shows widespread environment and human exposures. A phage clone designated L7-1 that selectively binds to imidacloprid was selected from a commercial phage display library containing linear 7-mer randomized amino acid residues. Using the clone L7-1, a competitive enzyme-linked immunosorbent assay (ELISA) for imidacloprid was developed. The half-maximum signal inhibition concentration (IC50) and the limit of detection (LOD) of the phage ELISA for imidacloprid were 96 and 2.3 ng ml(-1), respectively. This phage ELISA showed relatively low cross-reactivity with all of the tested compounds structurally similar to imidacloprid, less than 2% with the exception of 6-chloronicotinic acid, a metabolite of imidacloprid that showed 11.5%. The average recoveries of the phage ELISA for imidacloprid in water and soil samples were in the ranges of 74.6 to 86.3% and 72.5 to 93.6%, respectively. The results of the competitive phage ELISA for imidacloprid in the fortified samples agreed well with those of a high-performance liquid chromatography (HPLC) method. The simple phage-displayed peptide technology has been proven to be a convenient and efficient method for the development of an alternative format of ELISA for small molecules. Copyright © 2015 Elsevier Inc. All rights reserved.

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase.

PubMed

Gasparian, Marine E; Bobik, Tatyana V; Kim, Yana V; Ponomarenko, Natalia A; Dolgikh, Dmitry A; Gabibov, Alexander G; Kirpichnikov, Mikhail P

2013-11-01

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

In vitro high throughput phage display selection of ovarian cancer avid phage clones for near-infrared optical imaging.

PubMed

Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L

2014-01-01

Ovarian cancer is among the leading causes of cancer deaths in women, and is the most fatal gynecological malignancy. Poor outcomes of the disease are a direct result of inadequate detection and diagnostic methods, which may be overcome by the development of novel efficacious screening modalities. However, the advancement of such technologies is often time-consuming and costly. To overcome this hurdle, our laboratory has established a time and cost effective method of selecting and identifying ovarian carcinoma avid bacteriophage (phage) clones using high throughput phage display technology. These phage clones were selected from a filamentous phage fusion vector (fUSE5) 15-amino acid peptide library against human ovarian carcinoma (SKOV-3) cells, and identified by DNA sequencing. Two phage clones, pM6 and pM9, were shown to exhibit high binding affinity and specificity for SKOV-3 cells using micropanning, cell binding and fluorescent microscopy studies. To validate that the binding was mediated by the phage-displayed peptides, biotinylated peptides (M6 and M9) were synthesized and the specificity for ovarian carcinoma cells was analyzed. These results showed that M6 and M9 bound to SKOV-3 cells in a dose-response manner and exhibited EC50 values of 22.9 ± 2.0 μM and 12.2 ± 2.1μM (mean ± STD), respectively. Based on this, phage clones pM6 and pM9 were labeled with the near-infrared fluorophore AF680, and examined for their pharmacokinetic properties and tumor imaging abilities in vivo. Both phage successfully targeted and imaged SKOV-3 tumors in xenografted nude mice, demonstrating the ability of this method to quickly and cost effectively develop novel ovarian carcinoma avid phage.

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2

PubMed Central

Keresztessy, Zsolt; Csősz, Éva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N.; Lakey, Jeremy H.; Balajthy, Zoltán; Fésüs, László

2006-01-01

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q6, Q8, and Q22 are modified by TG2. Kinetic parameters of SnQ1 transamidation (KMapp = 250 μM, kcat = 18.3 sec−1, and kcat/KMapp = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research. PMID:17075129

Phage display selection of efficient glutamine-donor substrate peptides for transglutaminase 2.

PubMed

Keresztessy, Zsolt; Csosz, Eva; Hársfalvi, Jolán; Csomós, Krisztián; Gray, Joe; Lightowlers, Robert N; Lakey, Jeremy H; Balajthy, Zoltán; Fésüs, László

2006-11-01

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.

Lateral flow assay for rapid detection of white spot syndrome virus (WSSV) using a phage-displayed peptide as bio-recognition probe.

PubMed

Kulabhusan, Prabir Kumar; Rajwade, Jyutika M; Sahul Hameed, A S; Paknikar, Kishore M

2017-06-01

White spot disease caused by the white spot syndrome virus (WSSV) has a major socio-economic impact on shrimp farming in India. It has been realized that a field-usable diagnostic capable of rapid detection of WSSV can prevent huge economic losses in disease outbreaks. In this work, we explored the possibility of using a peptide as bio-recognition probe in a field-usable device for the detection of WSSV from infected shrimps and prawns. A commercially available random phage-display library was screened against rVP28 (a major structural protein of WSSV, expressed as a recombinant protein in Escherichia coli). A bacteriophage clone VP28-4L was obtained, and its binding to purified rVP28 protein as well as WSSV from infected shrimp Litopaeneus vannamei tissue was confirmed by ELISA and western blot. The apparent equilibrium dissociation constant (K d ,app) was calculated to be 810 nM. VP28-4L did not show cross-reactivity with any other shrimp viruses. A 12-mer peptide (pep28, with the sequence 'TFQAFDLSPFPS') displayed on the VP28-4L was synthesized, and its diagnostic potential was evaluated in a lateral flow assay (LFA). Visual detection of WSSV could be achieved using biotinylated-pep28 and streptavidin-conjugated gold nanoparticles. In LFA, 12.5 μg/mL of the virus could be detected from L. vannamei gill tissue homogenate within 20 min. Pep28 thus becomes an attractive candidate in bio-recognition of WSSV in field-usable diagnostic platforms benefitting the aquaculture sector.

Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

PubMed Central

Wu, You-Qiang; Qu, Hongchang; Sfyroera, Georgia; Tzekou, Apostolia; Kay, Brian K.; Nilsson, Bo; Ekdahl, Kristina Nilsson; Ricklin, Daniel; Lambris, John D.

2011-01-01

Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5–18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility. PMID:21339361

Molecular biomimetics: utilizing nature's molecular ways in practical engineering.

PubMed

Tamerler, Candan; Sarikaya, Mehmet

2007-05-01

In nature, proteins are the machinery that accomplish many functions through their specific recognition and interactions in biological systems from single-celled to multicellular organisms. Biomolecule-material interaction is accomplished via molecular specificity, leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, molecular recognition and, consequently, functions developed through successive cycles of mutation and selection. Using biology as a guide, we can now understand, engineer and control peptide-material interactions and exploit these to tailor novel materials and systems for practical applications. We adapted combinatorial biology protocols to display peptide libraries, either on the cell surface or on phages, to select short peptides specific to a variety of practical materials systems. Following the selection step, we determined the kinetics and stability of peptide binding experimentally to understand the bound peptide structure via modeling and its assembly via atomic force microscopy. The peptides were further engineered to have multiple repeats or their amino acid sequences varied to tailor their function. Both nanoparticles and flat inorganic substrates containing multimaterials patterned at the nano- and microscales were used for self-directed immobilization of molecular constructs. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems with wide ranging applications, from tissue engineering, drug delivery and biosensors, to nanotechnology and bioremediation. Here we give examples of protein-mediated functional materials in biology, peptide selection and engineering with affinity to inorganics, demonstrate potential utilizations in materials science, engineering and medicine, and describe future prospects.

Bio-recognition and functional lipidomics by glycosphingolipid transfer technology

PubMed Central

TAKI, Takao

2013-01-01

Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced. PMID:23883610

Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL.

PubMed

Schieferdecker, Aneta; Voigt, Mareike; Riecken, Kristoffer; Braig, Friederike; Schinke, Thorsten; Loges, Sonja; Bokemeyer, Carsten; Fehse, Boris; Binder, Mascha

2014-08-30

Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

Identification of GAPDH on the surface of Plasmodium sporozoites as a new candidate for targeting malaria liver invasion

PubMed Central

Kim, Min-Sik

2016-01-01

Malaria transmission begins when an infected mosquito delivers Plasmodium sporozoites into the skin. The sporozoite subsequently enters the circulation and infects the liver by preferentially traversing Kupffer cells, a macrophage-like component of the liver sinusoidal lining. By screening a phage display library, we previously identified a peptide designated P39 that binds to CD68 on the surface of Kupffer cells and blocks sporozoite traversal. In this study, we show that the P39 peptide is a structural mimic of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the sporozoite surface and that GAPDH directly interacts with CD68 on the Kupffer cell surface. Importantly, an anti-P39 antibody significantly inhibits sporozoite liver invasion without cross-reacting with mammalian GAPDH. Therefore, Plasmodium-specific GAPDH epitopes may provide novel antigens for the development of a prehepatic vaccine. PMID:27551151

Identification of GAPDH on the surface of Plasmodium sporozoites as a new candidate for targeting malaria liver invasion.

PubMed

Cha, Sung-Jae; Kim, Min-Sik; Pandey, Akhilesh; Jacobs-Lorena, Marcelo

2016-09-19

Malaria transmission begins when an infected mosquito delivers Plasmodium sporozoites into the skin. The sporozoite subsequently enters the circulation and infects the liver by preferentially traversing Kupffer cells, a macrophage-like component of the liver sinusoidal lining. By screening a phage display library, we previously identified a peptide designated P39 that binds to CD68 on the surface of Kupffer cells and blocks sporozoite traversal. In this study, we show that the P39 peptide is a structural mimic of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on the sporozoite surface and that GAPDH directly interacts with CD68 on the Kupffer cell surface. Importantly, an anti-P39 antibody significantly inhibits sporozoite liver invasion without cross-reacting with mammalian GAPDH. Therefore, Plasmodium-specific GAPDH epitopes may provide novel antigens for the development of a prehepatic vaccine. © 2016 Cha et al.

Effect of protein properties on display efficiency using the M13 phage display system.

PubMed

Imai, S; Mukai, Y; Takeda, T; Abe, Y; Nagano, K; Kamada, H; Nakagawa, S; Tsunoda, S; Tsutsumi, Y

2008-10-01

The M13 phage display system is a powerful technology for engineering proteins such as functional mutant proteins and peptides. In this system, it is necessary that the protein is displayed on the phage surface. Therefore, its application is often limited when a protein is poorly displayed. In this study, we attempted to understand the relationship between a protein's properties and its display efficiency using the well-known pIII and pVIII type phage display system. The display of positively charged SV40 NLS and HIV-1 Tat peptides on pill was less efficient than that of the neutrally charged RGDS peptide. When different molecular weight proteins (1.5-58 kDa) were displayed on pIII and pVIII, their display efficiencies were directly influenced by their molecular weights. These results indicate the usefulness in predicting a desired protein's compatibility with protein and peptide engineering using the phage display system.

The mzqLibrary--An open source Java library supporting the HUPO-PSI quantitative proteomics standard.

PubMed

Qi, Da; Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R

2015-09-01

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/. © 2015 The Authors. PROTEOMICS Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening and Identification of a Phage Display Derived Peptide That Specifically Binds to the CD44 Protein Region Encoded by Variable Exons.

PubMed

Zhang, Dan; Jia, Huan; Li, Weiming; Hou, Yingchun; Lu, Shaoying; He, Shuixiang

2016-01-01

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy. © 2015 Society for Laboratory Automation and Screening.

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica

PubMed Central

Wong, Ka H.; Tan, Wei Liang; Kini, Shruthi G.; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P.

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40–41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics. PMID:28680440

Vaccatides: Antifungal Glutamine-Rich Hevein-Like Peptides from Vaccaria hispanica.

PubMed

Wong, Ka H; Tan, Wei Liang; Kini, Shruthi G; Xiao, Tianshu; Serra, Aida; Sze, Sui Kwan; Tam, James P

2017-01-01

Hevein and hevein-like peptides are disulfide-constrained chitin-binding cysteine-rich peptides. They are divided into three subfamilies, 6C-, 8C-, and 10C-hevein-like peptides, based on the number of cysteine residues. In addition, hevein-like peptides can exist in two forms, short and long. The long C-terminal form found in hevein and 10C-hevein-like peptides contain a C-terminal protein cargo. In contrast, the short form without a protein cargo is found in all three subfamilies. Here, we report the discovery and characterization of two novel glutamine-rich and protein cargo-free 8C-hevein-like peptides, vaccatides vH1 and vH2, from Vaccaria hispanica of the Caryophyllaceae family. Proteomic analyses showed that the vaccatides are 40-41 amino acids in length and contain a chitin-binding domain. NMR determination revealed that vaccatide vH2 displays a highly compact structure with a N-terminal cystine knot and an addition C-terminal disulfide bond. Stability studies showed that this compact structure renders vaccatide vH2 resistant to thermal, chemical and proteolytic degradation. The chitin-binding vH2 was shown to inhibit the mycelium growth of four phyto-pathogenic fungal strains with IC 50 values in the micromolar range. Our findings show that vaccatides represent a new family of 8C-hevein-like peptides, which are protein cargo-free and glutamine-rich, characteristics that differentiate them from the prototypic hevein and the 10C-hevein-like peptides. In summary, this study enriches the existing library of hevein-like peptides and provides insight into their molecular diversity in sequence, structure and biosynthesis. Additionally, their highly disulfide-constrained structure could be used as a scaffold for developing metabolically and orally active peptidyl therapeutics.

Quantitation of the phosphoproteome using the library-assisted extracted ion chromatogram (LAXIC) strategy.

PubMed

Arrington, Justine V; Xue, Liang; Tao, W Andy

2014-01-01

Phosphorylation is a key posttranslational modification that regulates many signaling pathways, but quantifying changes in phosphorylation between samples can be challenging due to its low stoichiometry within cells. We have introduced a mass spectrometry-based label-free quantitation strategy termed LAXIC for the analysis of the phosphoproteome. This method uses a spiked-in synthetic peptide library designed to elute across the entire chromatogram for local normalization of phosphopeptides within complex samples. Normalization of phosphopeptides by library peptides that co-elute within a small time frame accounts for fluctuating ion suppression effects, allowing more accurate quantitation even when LC-MS performance varies. Here we explain the premise of LAXIC, the design of a suitable peptide library, and how the LAXIC algorithm can be implemented with software developed in-house.

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

PubMed Central

Szczyglowski, K; Hamburger, D; Kapranov, P; de Bruijn, F J

1997-01-01

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation. PMID:9276951

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

Development of bisphenol A-removing recombinant Escherichia coli by monomeric and dimeric surface display of bisphenol A-binding peptide.

PubMed

Maruthamuthu, Murali Kannan; Hong, Jiyeon; Arulsamy, Kulandaisamy; Somasundaram, Sivachandiran; Hong, SoonHo; Choe, Woo-Seok; Yoo, Ik-Keun

2018-04-01

Peptide-displaying Escherichia coli cells were investigated for use in adsorptive removal of bisphenol A (BPA) both in Luria-Bertani medium including BPA or ATM thermal paper eluted wastewater. Two recombinant strains were constructed with monomeric and dimeric repeats of the 7-mer BPA-binding peptide (KSLENSY), respectively. Greater than threefold increased adsorption of BPA [230.4 µmol BPA per g dry cell weight (DCW)] was found in dimeric peptide-displaying cells compared to monomeric strains (63.4 µmol per g DCW) in 15 ppm BPA solution. The selective removal of BPA from a mixture of BPA analogs (bisphenol F and bisphenol S) was verified in both monomeric and dimeric peptide-displaying cells. The binding chemistry of BPA with the peptide was assumed, based on molecular docking analysis, to be the interaction of BPA with serine and asparagine residues within the 7-mer peptide sequence. The peptide-displaying cells also functioned efficiently in thermal paper eluted wastewater containing 14.5 ppm BPA.

Development of a dedicated peptide tandem mass spectral library for conservation science.

PubMed

Fremout, Wim; Dhaenens, Maarten; Saverwyns, Steven; Sanyova, Jana; Vandenabeele, Peter; Deforce, Dieter; Moens, Luc

2012-05-30

In recent years, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art. Copyright © 2012 Elsevier B.V. All rights reserved.

Construction of a cDNA library for sea cucumber Acaudina leucoprocta and differential expression of ferritin peptide

NASA Astrophysics Data System (ADS)

Zhou, Jun; Hou, Fujing; Li, Ye; Su, Xiurong; Li, Taiwu; Jin, Chunhua

2016-07-01

Acaudina leucoprocta is an edible sea cucumber of economic interest that is widely distributed in China. Little information is available concerning the molecular genetics of this species although such knowledge would contribute to a better understanding of the optimal conditions for its aquaculture and its mechanisms of defense against disease. Therefore, we constructed a cDNA library and, based on bioinformatics analysis of the sequences, the functions of 75% of the cDNAs were identified, including those involved in cell structure, energy metabolism, mitochondrial function, and signal transduction pathways. Approximately 25% of genes in the library were unmatched. The gene for A. leucoprocta ferritin was also cloned. The predicted amino-acid sequence of ferritin displayed significant homology with other sea-cucumber counterparts but indicated that it was a new member of the ferritin family. Semiquantitative real-time RT-PCR indicated the highest levels of ferritin mRNA expression in the intestine. A polyclonal antibody of ferritin was also produced. These data provide a set of molecular tools essential for further studies of the functions of ferritin protein in A. leucoprocta.

Advances in phage display technology for drug discovery.

PubMed

Omidfar, Kobra; Daneshpour, Maryam

2015-06-01

Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery. This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs. Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

NIST Libraries of Peptide Fragmentation Mass Spectra Databass

National Institute of Standards and Technology Data Gateway

SRD 4 NIST Libraries of Peptide Fragmentation Mass Spectra Databass (PC database for purchase)   Interactive computer program for predicting thermodynamic and transport properties of pure fluids and fluid mixtures containing up to 20 components. The components are selected from a database of 196 components, mostly hydrocarbons.

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

PubMed

Zhao, Y; Gran, B; Pinilla, C; Markovic-Plese, S; Hemmer, B; Tzou, A; Whitney, L W; Biddison, W E; Martin, R; Simon, R

2001-08-15

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

A Review on Recent Patents and Applications of Inorganic Material Binding Peptides.

PubMed

Thota, Veeranjaneyulu; Perry, Carole C

2017-01-01

Although the popularity of using combinatorial display techniques for recognising unique peptides having high affinity for inorganic (nano) particles has grown rapidly, there are no systematic reviews showcasing current developments or patents on binding peptides specific to these materials. In this review, we summarize and discuss recent progress in patents on material binding peptides specifically exploring inorganic nano surfaces such as metals, metal oxides, minerals, carbonbased materials, polymer based materials, magnetic materials and semiconductors. We consider both the peptide display strategies used and the exploitation of the identified peptides in the generation of advanced nanomaterials. In order to get a clear picture on the number of patents and literature present to date relevant to inorganic material binding biomolecules and their applications, a thorough online search was conducted using national and worldwide databases. The literature search include standard bibliographic databases while patents included EPO Espacenet, WIPO patent scope, USPTO, Google patent search, Patent lens, etc. along with commercial databases such as Derwent and Patbase. Both English and American spellings were included in the searches. The initial number of patents found related to material binders were 981. After reading and excluding irrelevant patents such as organic binding peptides, works published before 2001, repeated patents, documents not in English etc., 51 highly relevant patents published from 2001 onwards were selected and analysed. These patents were further separated into six categories based on their target inorganic material and combinatorial library used. They include relevant patents on metal, metal oxide or combination binding peptides (19), magnetic and semiconductor binding peptides (8), carbon based (3), mineral (5), polymer (8) and other binders (9). Further, how these material specific binders have been used to synthesize simple to complex bio- or nano-materials, mediate the controlled biomineralization process, direct self-assembly and nanofabrication of ordered structures, facilitate the immobilization of functional biomolecules and construct inorganic-inorganic or organic-inorganic nano hybrids are concisely described. From analysis of recent literature and patents, we clearly show that biomimetic material binders are in the vanguard of new design approaches for novel nanomaterials with improved/ controlled physical and chemical properties that have no adverse effect on the structural or functional activities of the nanomaterials themselves. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Specific peptide for functionalization of GaN

NASA Astrophysics Data System (ADS)

Estephan, E.; Larroque, C.; Cloitre, T.; Cuisinier, F. J. G.; Gergely, C.

2008-04-01

Nanobiotechnology aims to exploit biomolecular recognition and self-assembly capabilities for integrating advanced materials into medicine and biology. However frequent problems are encountered at the interface of substrate-biological molecule, as the direct physical adsorption of biological molecules is dependent of unpredictable non-specific interactions with the surface, often causing their denaturation. Therefore, a proper functionalization of the substrate should avoid a loss of biological activity. In this work we address the functionalization of the semiconductor GaN (0001) for biosensing applications. The basic interest of using III-V class semiconductors is their good light emitting properties and a fair chemical stability that allows various applications of these materials. The technology chosen to elaborate GaN-specific peptides is the combinatorial phage-display method, a biological screening procedure based on affinity selection. An M13 bacteriophage library has been used to screen 10 10 different peptides against the GaN (0001) surface to finally isolate one specific peptide. The preferential attachment of the biotinylated selected peptide onto the GaN (0001), in close proximity to a surface of different chemical and structural composition has been demonstrated by fluorescence microscopy. Further physicochemical studies have been initiated to evaluate the semiconductor-peptide interface and understand the details in the specific recognition of peptides for semiconductor substrates. Fourier Transform Infrared spectroscopy in Attenuated Total Reflection mode (FTIR-ATR) has been employed to prove the presence of peptides on the surface. Our Atomic Force Microscopy (AFM) studies on the morphology of the GaN surface after functionalization revealed a total surface coverage by a very thin, homogeneous peptide layer. Due to its good biocompatibility, functionalized GaN devices might evolve in a new class of implantable biosensors for medical applications.

Toward a new and noninvasive diagnostic method of papillary thyroid cancer by using peptide vectorized contrast agents targeted to galectin-1.

PubMed

Fanfone, Deborah; Despretz, Nadège; Stanicki, Dimitri; Rubio-Magnieto, Jenifer; Fossépré, Mathieu; Surin, Mathieu; Rorive, Sandrine; Salmon, Isabelle; Vander Elst, Luce; Laurent, Sophie; Muller, Robert N; Saussez, Sven; Burtea, Carmen

2017-10-06

The incidence of papillary thyroid cancer has increased these last decades due to a better detection. High prevalence of nodules combined with the low incidence of thyroid cancers constitutes an important diagnostic challenge. We propose to develop an alternative diagnostic method to reduce the number of useless and painful thyroidectomies using a vectorized contrast agent for magnetic resonance imaging. Galectin-1 (gal-1), a protein overexpressed in well-differentiated thyroid cancer, has been targeted with a randomized linear 12-mer peptide library using the phage display technique. Selected peptides have been conjugated to ultrasmall superparamagnetic particles of iron oxide (USPIO). Peptides and their corresponding contrast agents have been tested in vitro for their specific binding and toxicity. Two peptides (P1 and P7) were selected according to their affinity toward gal-1. Their binding has been revealed by immunohistochemistry on human thyroid cancer biopsies, and they were co-localized with gal-1 by immunofluorescence on TPC-1 cell line. Both peptides induce a decrease in TPC-1 cells' adhesion to gal-1 immobilized on culture plates. After coupling to USPIO, the peptides preserved their affinity toward gal-1. Their specific binding has been corroborated by co-localization with gal-1 expressed by TPC-1 cells and by their ability to compete with anti-gal-1 antibody. The peptides and their USPIO derivatives produce no toxicity in HepaRG cells as determined by MTT assay. The vectorized contrast agents are potential imaging probes for thyroid cancer diagnosis. Moreover, the two gal-1-targeted peptides prevent cancer cell adhesion by interacting with the carbohydrate-recognition domain of gal-1.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

PubMed

Khlusevich, Yana; Matveev, Andrey; Baykov, Ivan; Bulychev, Leonid; Bormotov, Nikolai; Ilyichev, Ivan; Shevelev, Georgiy; Morozova, Vera; Pyshnyi, Dmitrii; Tikunova, Nina

2018-04-01

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies. Copyright © 2018. Published by Elsevier B.V.

Decreasing oxidative stress and neuroinflammation with a multifunctional peptide rescues memory deficits in mice with Alzheimer disease.

PubMed

Zhou, Wei-wei; Lu, Shuai; Su, Ya-jing; Xue, Di; Yu, Xiao-lin; Wang, Shao-wei; Zhang, He; Xu, Peng-xin; Xie, Xi-xiu; Liu, Rui-tian

2014-09-01

Alzheimer disease (AD) is characterized by extracellular senile plaques, intracellular neurofibrillary tangles, and memory loss. Aggregated amyloid-β (Aβ), oxidative stress, and inflammation have pivotal roles in the pathogenesis of AD. Therefore, the inhibition of Aβ-induced neurotoxicity, oxidative stress, and inflammation is a potential therapeutic strategy for the treatment of AD. In this study, a heptapeptide, isolated from a Ph.D.-C7C library by phage display, attenuated Aβ42-induced cytotoxicity in SH-SY5Y neuroblastoma cells and reduced Aβ42-induced oxidative stress by decreasing the production of reactive oxygen species and glutathione disulfide. As a result, glutathione level increased and superoxide dismutase and glutathione peroxidase activities were enhanced in vitro and in vivo. This peptide also suppressed the inflammatory response by decreasing the release of proinflammatory cytokines, such as tumor necrosis factor α and interleukin 1β, in microglia and by reducing microgliosis and astrogliosis in AD transgenic mice. This peptide was intracerebroventricularly administered to APPswe/PS1dE9 transgenic mice. We found that this peptide significantly improved spatial memory and reduced the amyloid plaque burden and soluble and insoluble Aβ levels. Our findings suggest that this multifunctional peptide has therapeutic potential for an Aβ-targeted treatment of AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

PubMed

Rahbarizadeh, F; Rasaee, M J; Forouzandeh Moghadam, M; Allameh, A A; Sadroddiny, E

2004-06-01

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer.

PubMed

Wendel, Ulrika; Persson, Nina; Risinger, Christian; Bengtsson, Eva; Nodin, Björn; Danielsson, Lena; Welinder, Charlotte; Nordin Fredrikson, Gunilla; Jansson, Bo; Blixt, Ola

2018-01-01

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer

PubMed Central

Wendel, Ulrika; Persson, Nina; Risinger, Christian; Bengtsson, Eva; Nodin, Björn; Danielsson, Lena; Welinder, Charlotte; Nordin Fredrikson, Gunilla

2018-01-01

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE. PMID:29420566

Peptide ligands targeting integrin alpha3beta1 in non-small cell lung cancer.

PubMed

Lau, Derick; Guo, Linlang; Liu, Ruiwu; Marik, Jan; Lam, Kit

2006-06-01

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.

Anti-tumor effects of a novel chimeric peptide on S180 and H22 xenografts bearing nude mice.

PubMed

Wu, Dongdong; Gao, Yanfeng; Chen, Lixiang; Qi, Yuanming; Kang, Qiaozhen; Wang, Haili; Zhu, Linyu; Ye, Yong; Zhai, Mingxia

2010-05-01

In recent years, many endogenous peptides have been identified by screening combinatory phage display peptide library, which play important roles in the process of angiogenesis. A heptapeptide, ATWLPPR, binds specifically to NRP-1 and selectively inhibits VEGF165 binding to VEGFR-2. Another heptapeptide, NLLMAAS, blocks both Ang-1 and Ang-2 binding to Tie-2 in a dose-dependent manner. In the present study, we aimed to connect ATWLPPR (V1) with NLLMAAS (V2) via a flexible linker, Ala-Ala, to reconstruct a novel peptide ATWLPPRAANLLMAAS (V3). We firstly investigated the anti-tumor and anti-angiogenic effects of peptide V3 on sarcoma S180 and hepatoma H22 bearing BALB/c nude mice. Mice were continuously subcutaneously administrated with normal saline, V1 (320microg/kg/d), V2 (320microg/kg/d), V1+V2 (320microg/kg/d), and V3 (160, 320 and 480microg/kg/d), for 7 days. Treatment with peptide V3 could significantly reduce the tumor weight and volume. Pathological examination showed that the tumors treated with peptide V3 had a larger region of necrosis than that of peptide V1, V2, and V1+V2 at the same dose. A significant decrease of microvessel density (MVD) in a dose-dependent manner was observed in each group of peptide V3. The results of pathological examination on normal tissue, lung, heart, liver, spleen, kidney and white blood cells showed that peptide V3 might have no significant toxicity. In conclusion, our results demonstrated that peptide V3 could be more effective on inhibiting tumor growth and angiogenesis than that of V1, V2, and V1+V2. Peptide V3 could be considered as a novel chimeric peptide with potent anti-tumor activity. Copyright (c) 2010 Elsevier Inc. All rights reserved.

In silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem-Cell Transplant Donors and Recipients: Understanding the Quantitative Immunobiology of Allogeneic Transplantation

PubMed Central

Jameson-Lee, Max; Koparde, Vishal; Griffith, Phil; Scalora, Allison F.; Sampson, Juliana K.; Khalid, Haniya; Sheth, Nihar U.; Batalo, Michael; Serrano, Myrna G.; Roberts, Catherine H.; Hess, Michael L.; Buck, Gregory A.; Neale, Michael C.; Manjili, Masoud H.; Toor, Amir Ahmed

2014-01-01

Donor T-cell mediated graft versus host (GVH) effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the human leukocyte antigen (HLA) molecules in each donor–recipient pair undergoing stem-cell transplantation (SCT). Whole exome sequencing has previously demonstrated a large number of non-synonymous single nucleotide polymorphisms (SNP) present in HLA-matched recipients of SCT donors (GVH direction). The nucleotide sequence flanking each of these SNPs was obtained and the amino acid sequence determined. All the possible nonameric peptides incorporating the variant amino acid resulting from these SNPs were interrogated in silico for their likelihood to be presented by the HLA class I molecules using the Immune Epitope Database stabilized matrix method (SMM) and NetMHCpan algorithms. The SMM algorithm predicted that a median of 18,396 peptides weakly bound HLA class I molecules in individual SCT recipients, and 2,254 peptides displayed strong binding. A similar library of presented peptides was identified when the data were interrogated using the NetMHCpan algorithm. The bioinformatic algorithm presented here demonstrates that there may be a high level of mHA variation in HLA-matched individuals, constituting a HLA-specific alloreactivity potential. PMID:25414699

In silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem-Cell Transplant Donors and Recipients: Understanding the Quantitative Immunobiology of Allogeneic Transplantation.

PubMed

Jameson-Lee, Max; Koparde, Vishal; Griffith, Phil; Scalora, Allison F; Sampson, Juliana K; Khalid, Haniya; Sheth, Nihar U; Batalo, Michael; Serrano, Myrna G; Roberts, Catherine H; Hess, Michael L; Buck, Gregory A; Neale, Michael C; Manjili, Masoud H; Toor, Amir Ahmed

2014-01-01

Donor T-cell mediated graft versus host (GVH) effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the human leukocyte antigen (HLA) molecules in each donor-recipient pair undergoing stem-cell transplantation (SCT). Whole exome sequencing has previously demonstrated a large number of non-synonymous single nucleotide polymorphisms (SNP) present in HLA-matched recipients of SCT donors (GVH direction). The nucleotide sequence flanking each of these SNPs was obtained and the amino acid sequence determined. All the possible nonameric peptides incorporating the variant amino acid resulting from these SNPs were interrogated in silico for their likelihood to be presented by the HLA class I molecules using the Immune Epitope Database stabilized matrix method (SMM) and NetMHCpan algorithms. The SMM algorithm predicted that a median of 18,396 peptides weakly bound HLA class I molecules in individual SCT recipients, and 2,254 peptides displayed strong binding. A similar library of presented peptides was identified when the data were interrogated using the NetMHCpan algorithm. The bioinformatic algorithm presented here demonstrates that there may be a high level of mHA variation in HLA-matched individuals, constituting a HLA-specific alloreactivity potential.

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen

PubMed Central

Bobes, Raúl J.; Navarrete-Perea, José; Ochoa-Leyva, Adrián; Anaya, Víctor Hugo; Hernández, Marisela; Cervantes-Torres, Jacquelynne; Estrada, Karel; Sánchez-Lopez, Filiberto; Soberón, Xavier; Rosas, Gabriela; Nunes, Cáris Maroni; García-Varela, Martín; Sotelo-Mundo, Rogerio Rafael; López-Zavala, Alonso Alexis; Gevorkian, Goar; Acero, Gonzalo; Laclette, Juan P.; Fragoso, Gladis

2017-01-01

ABSTRACT Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps. Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines. PMID:28923896

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen.

PubMed

Bobes, Raúl J; Navarrete-Perea, José; Ochoa-Leyva, Adrián; Anaya, Víctor Hugo; Hernández, Marisela; Cervantes-Torres, Jacquelynne; Estrada, Karel; Sánchez-Lopez, Filiberto; Soberón, Xavier; Rosas, Gabriela; Nunes, Cáris Maroni; García-Varela, Martín; Sotelo-Mundo, Rogerio Rafael; López-Zavala, Alonso Alexis; Gevorkian, Goar; Acero, Gonzalo; Laclette, Juan P; Fragoso, Gladis; Sciutto, Edda

2017-12-01

Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines. Copyright © 2017 American Society for Microbiology.

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

PubMed

Egami, Yoko; Narushima, Yuta; Ohshima, Motohiro; Yoshida, Akira; Yoneta, Naruki; Masaki, Yasufumi; Itoh, Kunihiko

2018-01-01

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

De novo designed library of linear helical peptides: an exploratory tool in the discovery of protein-protein interaction modulators.

PubMed

Bonache, M Ángeles; Balsera, Beatriz; López-Méndez, Blanca; Millet, Oscar; Brancaccio, Diego; Gómez-Monterrey, Isabel; Carotenuto, Alfonso; Pavone, Luigi M; Reille-Seroussi, Marie; Gagey-Eilstein, Nathalie; Vidal, Michel; de la Torre-Martinez, Roberto; Fernández-Carvajal, Asia; Ferrer-Montiel, Antonio; García-López, M Teresa; Martín-Martínez, Mercedes; de Vega, M Jesús Pérez; González-Muñiz, Rosario

2014-05-12

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

Enhancing the Breadth and Efficacy of Therapeutic Vaccines for Breast Cancer

DTIC Science & Technology

2014-10-01

us to generate full length recombinant TCR proteins that will be used to screen against the bacteriophage peptide library the Slansky/Kappler team has...the Denver team bacteriophage peptide library for HLA-A2 to screen these T cells. As a result we have abandoned this approach in order to focus our

Methods for Selecting Phage Display Antibody Libraries.

PubMed

Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

2016-01-01

The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Stimulating CTL Towards HER2/neu Overexpressing Breast Cancer

DTIC Science & Technology

2000-10-01

cytotoxicity possibly due to the peptides poor solubility and poor binding affinity. To gain further insight into the factors that govern CTL activity, we...662 peptide. Objective: Complete by 12/96. Methods: A soluble recombinant form of HLA-A2 is folded in vitro in the presence of j2m and HN654-662. The...decided to substitute the first position from isoleucine to lysine to improve solubility of the peptide library. The library was used in our in vitro

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

PubMed

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

PEP-on-DEP: A competitive peptide-based disposable electrochemical aptasensor for renin diagnostics.

PubMed

Biyani, Manish; Kawai, Keiko; Kitamura, Koichiro; Chikae, Miyuki; Biyani, Madhu; Ushijima, Hiromi; Tamiya, Eiichi; Yoneda, Takashi; Takamura, Yuzuru

2016-10-15

Antibody-based immunosensors are relatively less accessible to a wide variety of unreachable targets, such as low-molecular-weight biomarkers that represent a rich untapped source of disease-specific diagnostic information. Here, we present a peptide aptamer-based electrochemical sensor technology called 'PEP-on-DEP' to detect less accessible target molecules, such as renin, and to improve the quality of life. Peptide-based aptamers represent a relatively smart class of affinity binders and show great promise in biosensor development. Renin is involved in the regulation of arterial blood pressure and is an emerging biomarker protein for predicting cardiovascular risk and prognosis. To our knowledge, no studies have described aptamer molecules that can be used as new potent probes for renin. Here, we describe a portable electrochemical biosensor platform based on the newly identified peptide aptamer molecules for renin. We constructed a randomized octapeptide library pool with diversified sequences and selected renin specific peptide aptamers using cDNA display technology. We identified a few peptide aptamer sequences with a KD in the µM binding affinity range for renin. Next, we grafted the selected peptide aptamers onto gold nanoparticles and detected renin in a one-step competitive assay using our originally developed DEP (Disposable Electrochemical Printed) chip and a USB powered portable potentiostat system. We successfully detected renin in as little as 300ngmL(-1) using the PEP-on-DEP method. Thus, the generation and characterization of novel probes for unreachable target molecules by merging a newly identified peptide aptamer with electrochemical transduction allowed for the development of a more practical biosensor that, in principle, can be adapted to develop a portable, low-cost and mass-producible biosensor for point-of-care applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

PubMed

Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

2015-01-01

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Computational smart polymer design based on elastin protein mutability.

PubMed

Tarakanova, Anna; Huang, Wenwen; Weiss, Anthony S; Kaplan, David L; Buehler, Markus J

2017-05-01

Soluble elastin-like peptides (ELPs) can be engineered into a range of physical forms, from hydrogels and scaffolds to fibers and artificial tissues, finding numerous applications in medicine and engineering as "smart polymers". Elastin-like peptides are attractive candidates as a platform for novel biomaterial design because they exhibit a highly tunable response spectrum, with reversible phase transition capabilities. Here, we report the design of the first virtual library of elastin-like protein models using methods for enhanced sampling to study the effect of peptide chemistry, chain length, and salt concentration on the structural transitions of ELPs, exposing associated molecular mechanisms. We describe the behavior of the local molecular structure under increasing temperatures and the effect of peptide interactions with nearest hydration shell water molecules on peptide mobility and propensity to exhibit structural transitions. Shifts in the magnitude of structural transitions at the single-molecule scale are explained from the perspective of peptide-ion-water interactions in a library of four unique elastin-like peptide systems. Predictions of structural transitions are subsequently validated in experiment. This library is a valuable resource for recombinant protein design and synthesis as it elucidates mechanisms at the single-molecule level, paving a feedback path between simulation and experiment for smart material designs, with applications in biomedicine and diagnostic devices. Copyright © 2017. Published by Elsevier Ltd.

Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.

PubMed

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.

Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

PubMed Central

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

Phage as a template to grow bone mineral nanocrystals.

PubMed

Cao, Binrui; Xu, Hong; Mao, Chuanbin

2014-01-01

Phage display is a biotechnique that fuses functional peptides on the outer surface of filamentous phage by inserting DNA encoding the peptides into the genes of its coat proteins. The resultant peptide-displayed phage particles have been widely used as biotemplates for the synthesis of functional hybrid nanomaterials. Here, we describe the bioengineering of M13 filamentous phage to surface-display bone mineral (hydroxyapatite (HAP))-nucleating peptides derived from dentin matrix protein-1 and using the engineered phage as a biotemplate to grow HAP nanocrystals.

The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.

PubMed

Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Seppä, Titta; Akter, Sultana; Khan, Liton Md Ferdhos; Lamminmäki, Urpo

2014-09-19

Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins. In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection. The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.

Development of a multipurpose scaffold for the display of peptide loops

PubMed Central

Rossmann, Maxim; J. Greive, Sandra; Moschetti, Tommaso; Dinan, Michael

2017-01-01

Abstract Protein–protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization. When combined with the significant thermal stability of the RadA protein, these features create a versatile multipurpose scaffold system. Using various orthogonal biophysical techniques, we show that peptides displayed on the scaffold bind to their natural targets in a fashion similar to linear parent peptides. We use the examples of CK2β/CK2α kinase and TPX2/Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RAD scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts. PMID:28444399

Spectra library assisted de novo peptide sequencing for HCD and ETD spectra pairs.

PubMed

Yan, Yan; Zhang, Kaizhong

2016-12-23

De novo peptide sequencing via tandem mass spectrometry (MS/MS) has been developed rapidly in recent years. With the use of spectra pairs from the same peptide under different fragmentation modes, performance of de novo sequencing is greatly improved. Currently, with large amount of spectra sequenced everyday, spectra libraries containing tens of thousands of annotated experimental MS/MS spectra become available. These libraries provide information of the spectra properties, thus have the potential to be used with de novo sequencing to improve its performance. In this study, an improved de novo sequencing method assisted with spectra library is proposed. It uses spectra libraries as training datasets and introduces significant scores of the features used in our previous de novo sequencing method for HCD and ETD spectra pairs. Two pairs of HCD and ETD spectral datasets were used to test the performance of the proposed method and our previous method. The results show that this proposed method achieves better sequencing accuracy with higher ranked correct sequences and less computational time. This paper proposed an advanced de novo sequencing method for HCD and ETD spectra pair and used information from spectra libraries and significant improved previous similar methods.

Architectural Insight into Inovirus-Associated Vectors (IAVs) and Development of IAV-Based Vaccines Inducing Humoral and Cellular Responses: Implications in HIV-1 Vaccines

PubMed Central

Hassapis, Kyriakos A.; Stylianou, Dora C.; Kostrikis, Leondios G.

2014-01-01

Inovirus-associated vectors (IAVs) are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1. PMID:25525909

Architectural insight into inovirus-associated vectors (IAVs) and development of IAV-based vaccines inducing humoral and cellular responses: implications in HIV-1 vaccines.

PubMed

Hassapis, Kyriakos A; Stylianou, Dora C; Kostrikis, Leondios G

2014-12-17

Inovirus-associated vectors (IAVs) are engineered, non-lytic, filamentous bacteriophages that are assembled primarily from thousands of copies of the major coat protein gp8 and just five copies of each of the four minor coat proteins gp3, gp6, gp7 and gp9. Inovirus display studies have shown that the architecture of inoviruses makes all coat proteins of the inoviral particle accessible to the outside. This particular feature of IAVs allows foreign antigenic peptides to be displayed on the outer surface of the virion fused to its coat proteins and for more than two decades has been exploited in many applications including antibody or peptide display libraries, drug design, and vaccine development against infectious and non-infectious diseases. As vaccine carriers, IAVs have been shown to elicit both a cellular and humoral response against various pathogens through the display of antibody epitopes on their coat proteins. Despite their high immunogenicity, the goal of developing an effective vaccine against HIV-1 has not yet materialized. One possible limitation of previous efforts was the use of broadly neutralizing antibodies, which exhibited autoreactivity properties. In the past five years, however, new, more potent broadly neutralizing antibodies that do not exhibit autoreactivity properties have been isolated from HIV-1 infected individuals, suggesting that vaccination strategies aimed at producing such broadly neutralizing antibodies may confer protection against infection. The utilization of these new, broadly neutralizing antibodies in combination with the architectural traits of IAVs have driven the current developments in the design of an inovirus-based vaccine against HIV-1. This article reviews the applications of IAVs in vaccine development, with particular emphasis on the design of inoviral-based vaccines against HIV-1.

Discovery of Neuropeptides in the Nematode Ascaris suum by Database Mining and Tandem Mass Spectrometry

PubMed Central

Jarecki, Jessica L.; Frey, Brian L.; Smith, Lloyd M.; Stretton, Antony O.

2011-01-01

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs), or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1X coverage. PMID:21524146

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

GLCF: Library

Science.gov Websites

Global Land Cover Facility About GLCF Research Publications Data & Products Gallery Library Services Contact Site Map Go Library Documents Proposal Reports Publications FAQ Display Materials Release News Archive Library * Display Materials * Documents * News Archive * Software e-link 4321

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

PubMed Central

Polonelli, Luciano; Ciociola, Tecla; Magliani, Walter; Zanello, Pier Paolo; D'Adda, Tiziana; Galati, Serena; De Bernardis, Flavia; Arancia, Silvia; Gabrielli, Elena; Pericolini, Eva; Vecchiarelli, Anna; Arruda, Denise C.; Pinto, Marcia R.; Travassos, Luiz R.; Pertinhez, Thelma A.; Spisni, Alberto; Conti, Stefania

2012-01-01

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents. PMID:22470523

Raster graphics display library

NASA Technical Reports Server (NTRS)

Grimsrud, Anders; Stephenson, Michael B.

1987-01-01

The Raster Graphics Display Library (RGDL) is a high level subroutine package that give the advanced raster graphics display capabilities needed. The RGDL uses FORTRAN source code routines to build subroutines modular enough to use as stand-alone routines in a black box type of environment. Six examples are presented which will teach the use of RGDL in the fastest, most complete way possible. Routines within the display library that are used to produce raster graphics are presented in alphabetical order, each on a separate page. Each user-callable routine is described by function and calling parameters. All common blocks that are used in the display library are listed and the use of each variable within each common block is discussed. A reference on the include files that are necessary to compile the display library is contained. Each include file and its purpose are listed. The link map for MOVIE.BYU version 6, a general purpose computer graphics display system that uses RGDL software, is also contained.

Turn stability in beta-hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns.

PubMed

Blandl, Tamas; Cochran, Andrea G; Skelton, Nicholas J

2003-02-01

The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.

Role of molecular mimicry to HIV-1 peptides in HIV-1–related immunologic thrombocytopenia

PubMed Central

Li, Zongdong; Nardi, Michael A.; Karpatkin, Simon

2005-01-01

Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which antibody is directed against an immunodominant epitope of the β3 (glycoprotein IIIa [GPIIIa]) integrin, GPIIIa49-66. This antibody induces thrombocytopenia by a novel complement-independent mechanism in which platelets are fragmented by antibody-induced generation of H2O2 derived from the interaction of platelet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for antibody-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, using a filamentous phage display 7-mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Ten were molecular mimics with close sequence similarity to HIV-1 proteins nef, gag, env, and pol. Seven were synthesized as 10-mers from their known HIV-1 sequence and found to inhibit anti–GPIIIa49-66–induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a variant region of the protein. These data provide strong support for molecular mimicry in HIV-1-immunologic thrombocytopenia within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated. PMID:15774614

Biomolecular detection using a metal semiconductor field effect transistor

NASA Astrophysics Data System (ADS)

Estephan, Elias; Saab, Marie-Belle; Buzatu, Petre; Aulombard, Roger; Cuisinier, Frédéric J. G.; Gergely, Csilla; Cloitre, Thierry

2010-04-01

In this work, our attention was drawn towards developing affinity-based electrical biosensors, using a MESFET (Metal Semiconductor Field Effect Transistor). Semiconductor (SC) surfaces must be prepared before the incubations with biomolecules. The peptides route was adapted to exceed and bypass the limits revealed by other types of surface modification due to the unwanted unspecific interactions. As these peptides reveal specific recognition of materials, then controlled functionalization can be achieved. Peptides were produced by phage display technology using a library of M13 bacteriophage. After several rounds of bio-panning, the phages presenting affinities for GaAs SC were isolated; the DNA of these specific phages were sequenced, and the peptide with the highest affinity was synthesized and biotinylated. To explore the possibility of electrical detection, the MESFET fabricated with the GaAs SC were used to detect the streptavidin via the biotinylated peptide in the presence of the bovine Serum Albumin. After each surface modification step, the IDS (current between the drain and the source) of the transistor was measured and a decrease in the intensity was detected. Furthermore, fluorescent microscopy was used in order to prove the specificity of this peptide and the specific localisation of biomolecules. In conclusion, the feasibility of producing an electrical biosensor using a MESFET has been demonstrated. Controlled placement, specific localization and detection of biomolecules on a MESFET transistor were achieved without covering the drain and the source. This method of functionalization and detection can be of great utility for biosensing application opening a new way for developing bioFETs (Biomolecular Field-Effect Transistor).

A Label-Free Electrochemical Impedance Cytosensor Based on Specific Peptide-Fused Phage Selected from Landscape Phage Library

NASA Astrophysics Data System (ADS)

Han, Lei; Liu, Pei; Petrenko, Valery A.; Liu, Aihua

2016-02-01

One of the major challenges in the design of biosensors for cancer diagnosis is to introduce a low-cost and selective probe that can recognize cancer cells. In this paper, we combined the phage display technology and electrochemical impedance spectroscopy (EIS) to develop a label-free cytosensor for the detection of cancer cells, without complicated purification of recognition elements. Fabrication steps of the cytosensing interface were monitored by EIS. Due to the high specificity of the displayed octapeptides and avidity effect of their multicopy display on the phage scaffold, good biocompatibility of recombinant phage, the fibrous nanostructure of phage, and the inherent merits of EIS technology, the proposed cytosensor demonstrated a wide linear range (2.0 × 102 - 2.0 × 108 cells mL-1), a low limit of detection (79 cells mL-1, S/N = 3), high specificity, good inter-and intra-assay reproducibility and satisfactory storage stability. This novel cytosensor designing strategy will open a new prospect for rapid and label-free electrochemical platform for tumor diagnosis.

Specificity of Mimotope-Induced Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Antibodies Does Not Ensure Biological Activity

PubMed Central

Hofstetter, Gerlinde; Balazs, Nina; Smole, Ursula; Ferrone, Soldano; Scheiner, Otto; Breiteneder, Heimo; Pehamberger, Hubert; Wagner, Stefan

2011-01-01

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes. PMID:21573118

Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity.

PubMed

Latzka, Julia; Gaier, Sonja; Hofstetter, Gerlinde; Balazs, Nina; Smole, Ursula; Ferrone, Soldano; Scheiner, Otto; Breiteneder, Heimo; Pehamberger, Hubert; Wagner, Stefan

2011-05-06

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.

Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles.

PubMed

Yamane, Asaka; Fukui, Mina; Sugimura, Yoshiaki; Itoh, Miho; Alea, Mileidys Perez; Thomas, Vincent; El Alaoui, Said; Akiyama, Masashi; Hitomi, Kiyotaka

2010-09-01

Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

A RHAMM Mimetic Peptide Blocks Hyaluronan Signaling and Reduces Inflammation and Fibrogenesis in Excisional Skin Wounds

PubMed Central

Tolg, Cornelia; Hamilton, Sara R.; Zalinska, Ewa; McCulloch, Lori; Amin, Ripal; Akentieva, Natalia; Winnik, Francoise; Savani, Rashmin; Bagli, Darius J.; Luyt, Len G.; Cowman, Mary K.; McCarthy, Jim B.; Turley, Eva A.

2013-01-01

Hyaluronan is activated by fragmentation and controls inflammation and fibroplasia during wound repair and diseases (eg, cancer). Hyaluronan-binding peptides were identified that modify fibrogenesis during skin wound repair. Peptides were selected from 7- to 15mer phage display libraries by panning with hyaluronan-Sepharose beads and assayed for their ability to block fibroblast migration in response to hyaluronan oligosaccharides (10 kDa). A 15mer peptide (P15-1), with homology to receptor for hyaluronan mediated motility (RHAMM) hyaluronan binding sequences, was the most effective inhibitor. P15-1 bound to 10-kDa hyaluronan with an affinity of Kd = 10−7 and appeared to specifically mimic RHAMM since it significantly reduced binding of hyaluronan oligosaccharides to recombinant RHAMM but not to recombinant CD44 or TLR2,4, and altered wound repair in wild-type but not RHAMM−/− mice. One topical application of P15-1 to full-thickness excisional rat wounds significantly reduced wound macrophage number, fibroblast number, and blood vessel density compared to scrambled, negative control peptides. Wound collagen 1, transforming growth factor β-1, and α-smooth muscle actin were reduced, whereas tenascin C was increased, suggesting that P15-1 promoted a form of scarless healing. Signaling/microarray analyses showed that P15-1 blocks RHAMM-regulated focal adhesion kinase pathways in fibroblasts. These results identify a new class of reagents that attenuate proinflammatory, fibrotic repair by blocking hyaluronan oligosaccharide signaling. PMID:22889846

Synthesis and screening of one-bead-one-compound cyclic peptide libraries.

PubMed

Qian, Ziqing; Upadhyaya, Punit; Pei, Dehua

2015-01-01

Cyclic peptides have been a rich source of biologically active molecules. Herein we present a method for the combinatorial synthesis and screening of large one-bead-one-compound (OBOC) libraries of cyclic peptides against biological targets such as proteins. Up to ten million different cyclic peptides are rapidly synthesized on TentaGel microbeads by the split-and-pool synthesis method and subjected to a multistage screening protocol which includes magnetic sorting, on-bead enzyme-linked and fluorescence-based assays, and in-solution binding analysis of cyclic peptides selectively released from single beads by fluorescence anisotropy. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. This method allows a single researcher to synthesize and screen up to ten million cyclic peptides and identify the most active ligand(s) in ~1 month, without the time-consuming and expensive hit resynthesis or the use of any special equipment.

Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.

PubMed

Hu, Hongbo; Li, Li; Kao, Richard Y; Kou, Binbin; Wang, Zhanguo; Zhang, Liang; Zhang, Huiyuan; Hao, Zhiyong; Tsui, Wayne H; Ni, Anping; Cui, Lianxian; Fan, Baoxing; Guo, Feng; Rao, Shuan; Jiang, Chengyu; Li, Qian; Sun, Manji; He, Wei; Liu, Gang

2005-01-01

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.

Molecular biomimetics: GEPI-based biological routes to technology.

PubMed

Tamerler, Candan; Khatayevich, Dmitriy; Gungormus, Mustafa; Kacar, Turgay; Oren, E Emre; Hnilova, Marketa; Sarikaya, Mehmet

2010-01-01

In nature, the viability of biological systems is sustained via specific interactions among the tens of thousands of proteins, the major building blocks of organisms from the simplest single-celled to the most complex multicellular species. Biomolecule-material interaction is accomplished with molecular specificity and efficiency leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, Mother Nature developed molecular recognition by successive cycles of mutation and selection. Molecular specificity of probe-target interactions, e.g., ligand-receptor, antigen-antibody, is always based on specific peptide molecular recognition. Using biology as a guide, we can now understand, engineer, and control peptide-material interactions and exploit them as a new design tool for novel materials and systems. We adapted the protocols of combinatorially designed peptide libraries, via both cell surface or phage display methods; using these we select short peptides with specificity to a variety of practical materials. These genetically engineered peptides for inorganics (GEPI) are then studied experimentally to establish their binding kinetics and surface stability. The bound peptide structure and conformations are interrogated both experimentally and via modeling, and self-assembly characteristics are tested via atomic force microscopy. We further engineer the peptide binding and assembly characteristics using a computational biomimetics approach where bioinformatics based peptide-sequence similarity analysis is developed to design higher generation function-specific peptides. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems in a wide-range of applications from tissue engineering, disease diagnostics, and therapeutics to various areas of nanotechnology where integration is required among inorganic, organic and biological materials. Here, we describe lessons from biology with examples of protein-mediated functional biological materials, explain how novel peptides can be designed with specific affinity to inorganic solids using evolutionary engineering approaches, give examples of their potential utilizations in technology and medicine, and, finally, provide a summary of challenges and future prospects. (c) 2010 Wiley Periodicals, Inc.

Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd.

PubMed

Aoshi, Taiki; Suzuki, Mina; Uchijima, Masato; Nagata, Toshi; Koide, Yukio

2005-03-01

Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.

An antimicrobial bicyclic peptide from chemical space against multidrug resistant Gram-negative bacteria.

PubMed

Di Bonaventura, Ivan; Baeriswyl, Stéphane; Capecchi, Alice; Gan, Bee-Ha; Jin, Xian; Siriwardena, Thissa N; He, Runze; Köhler, Thilo; Pompilio, Arianna; Di Bonaventura, Giovanni; van Delden, Christian; Javor, Sacha; Reymond, Jean-Louis

2018-05-15

We used the concept of chemical space to explore a virtual library of bicyclic peptides formed by double thioether cyclization of a precursor linear peptide, and identified an antimicrobial bicyclic peptide (AMBP) with remarkable activity against several MDR strains of Acinetobacter baumannii and Pseudomonas aeruginosa.

Comprehensive computational design of ordered peptide macrocycles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hosseinzadeh, Parisa; Bhardwaj, Gaurav; Mulligan, Vikram Khipple

Mixed chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to-date, but there is currently no way to systematically search through the structural space spanned by such compounds for new drug candidates. Natural proteins do not provide a useful guide: peptide macrocycles lack regular secondary structures and hydrophobic cores and have different backbone torsional constraints. Hence the development of new peptide macrocycles has been approached by modifying natural products or using library selection methods; the former is limited by the small number of known structures, and the latter by the limited size and diversity accessible throughmore » library-based methods. To overcome these limitations, here we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L and D amino acids. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. We synthesize and characterize by NMR twelve 7-10 residue macrocycles, 9 of which have structures very close to the design models in solution. NMR structures of three 11-14 residue bicyclic designs are also very close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide based macrocycles unparalleled for other molecular systems, and vastly increase the available starting scaffolds for both rational drug design and library selection methods.« less

Management Matters. Display and Promotion Ideas for Library Media Centers

ERIC Educational Resources Information Center

Pappas, Marjorie L.

2005-01-01

School library media centers should be warm and inviting places where the environment entices children to read and explore. Creative bulletin board and case displays along with other exhibits help to make the library media center an exciting place. Bulletin board displays that promote authors, books, and exciting ideas motivate children to find…

Rapid discovery of peptide capture candidates with demonstrated specificity for structurally similar toxins

NASA Astrophysics Data System (ADS)

Sarkes, Deborah A.; Hurley, Margaret M.; Coppock, Matthew B.; Farrell, Mikella E.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

2016-05-01

Peptides have emerged as viable alternatives to antibodies for molecular-based sensing due to their similarity in recognition ability despite their relative structural simplicity. Various methods for peptide capture reagent discovery exist, including phage display, yeast display, and bacterial display. One of the primary advantages of peptide discovery by bacterial display technology is the speed to candidate peptide capture agent, due to both rapid growth of bacteria and direct utilization of the sorted cells displaying each individual peptide for the subsequent round of biopanning. We have previously isolated peptide affinity reagents towards protective antigen of Bacillus anthracis using a commercially available automated magnetic sorting platform with improved enrichment as compared to manual magnetic sorting. In this work, we focus on adapting our automated biopanning method to a more challenging sort, to demonstrate the specificity possible with peptide capture agents. This was achieved using non-toxic, recombinant variants of ricin and abrin, RiVax and abrax, respectively, which are structurally similar Type II ribosomal inactivating proteins with significant sequence homology. After only two rounds of biopanning, enrichment of peptide capture candidates binding abrax but not RiVax was achieved as demonstrated by Fluorescence Activated Cell Sorting (FACS) studies. Further sorting optimization included negative sorting against RiVax, proper selection of autoMACS programs for specific sorting rounds, and using freshly made buffer and freshly thawed protein target for each round of biopanning for continued enrichment over all four rounds. Most of the resulting candidates from biopanning for abrax binding peptides were able to bind abrax but not RiVax, demonstrating that short peptide sequences can be highly specific even at this early discovery stage.

Synthetic Molecular Evolution of Membrane-Active Peptides

NASA Astrophysics Data System (ADS)

Wimley, William

The physical chemistry of membrane partitioning largely determines the function of membrane active peptides. Membrane-active peptides have potential utility in many areas, including in the cellular delivery of polar compounds, cancer therapy, biosensor design, and in antibacterial, antiviral and antifungal therapies. Yet, despite decades of research on thousands of known examples, useful sequence-structure-function relationships are essentially unknown. Because peptide-membrane interactions within the highly fluid bilayer are dynamic and heterogeneous, accounts of mechanism are necessarily vague and descriptive, and have little predictive power. This creates a significant roadblock to advances in the field. We are bypassing that roadblock with synthetic molecular evolution: iterative peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, focused libraries using structural and biophysical principles to design the sequence space around the template. Orthogonal high-throughput screening is used to identify gain-of-function peptides by simultaneously selecting for several different properties (e.g. solubility, activity and toxicity). Multiple generations of iterative library design and screening have enabled the identification of membrane-active sequences with heretofore unknown properties, including clinically relevant, broad-spectrum activity against drug-resistant bacteria and enveloped viruses as well as pH-triggered macromolecular poration.

Enhanced Sensitive Immunoassay: Noncompetitive Phage Anti-Immune Complex Assay for the Determination of Malachite Green and Leucomalachite Green

PubMed Central

2015-01-01

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG–mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R2LMG = 0.9841; R2MG = 0.993; R2Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety. PMID:25077381

Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display.

PubMed

Patel, Chirag A; Wang, Jinqing; Wang, Xinwei; Dong, Feng; Zhong, Pingyu; Luo, Peter P; Wang, Kevin C

2011-09-01

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.

The mzqLibrary – An open source Java library supporting the HUPO‐PSI quantitative proteomics standard

PubMed Central

Zhang, Huaizhong; Fan, Jun; Perkins, Simon; Pisconti, Addolorata; Simpson, Deborah M.; Bessant, Conrad; Hubbard, Simon; Jones, Andrew R.

2015-01-01

The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML‐based format, capable of representing data about two‐dimensional features from LC‐MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java‐based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)‐level quantification values from peptide‐level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC‐MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in‐built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq‐lib/. PMID:26037908

A Combinatorial Platform for the Optimization of Peptidomimetic Methyl-Lysine Reader Antagonists

NASA Astrophysics Data System (ADS)

Barnash, Kimberly D.

Post-translational modification of histone N-terminal tails mediates chromatin compaction and, consequently, DNA replication, transcription, and repair. While numerous post-translational modifications decorate histone tails, lysine methylation is an abundant mark important for both gene activation and repression. Methyl-lysine (Kme) readers function through binding mono-, di-, or trimethyl-lysine. Chemical intervention of Kme readers faces numerous challenges due to the broad surface-groove interactions between readers and their cognate histone peptides; yet, the increasing interest in understanding chromatin-modifying complexes suggests tractable lead compounds for Kme readers are critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains and complexes. The successful discovery of a peptide-derived chemical probe, UNC3866, for the Polycomb repressive complex 1 (PRC1) chromodomain Kme readers has proven the potential for selective peptidomimetic inhibition of reader function. Unfortunately, the systematic modification of peptides-to-peptidomimetics is a costly and inefficient strategy for target-class hit discovery against Kme readers. Through the exploration of biased chemical space via combinatorial on-bead libraries, we have developed two concurrent methodologies for Kme reader chemical probe discovery. We employ biased peptide combinatorial libraries as a hit discovery strategy with subsequent optimization via iterative targeted libraries. Peptide-to-peptidomimetic optimization through targeted library design was applied based on structure-guided library design around the interaction of the endogenous peptide ligand with three target Kme readers. Efforts targeting the WD40 reader EED led to the discovery of the 3-mer peptidomimetic ligand UNC5115 while combinatorial repurposing of UNC3866 for off-target chromodomains resulted in the discovery of UNC4991, a CDYL/2-selective ligand, and UNC4848, a MPP8 and CDYL/2 ligand. Ultimately, our efforts demonstrate the generalizability of a peptidomimetic combinatorial platform for the optimization of Kme reader ligands in a target class manner.

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F.W.; Rosenberg, A.H.

1998-06-16

Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest. 1 fig.

Cytoplasmic bacteriophage display system

DOEpatents

Studier, F. William; Rosenberg, Alan H.

1998-06-16

Disclosed are display vectors comprising DNA encoding a portion of a structural protein from a cytoplasmic bacteriophage, joined covalently to a protein or peptide of interest. Exemplified are display vectors wherein the structural protein is the T7 bacteriophage capsid protein. More specifically, in the exemplified display vectors the C-terminal amino acid residue of the portion of the capsid protein is joined to the N-terminal residue of the protein or peptide of interest. The portion of the T7 capsid protein exemplified comprises an N-terminal portion corresponding to form 10B of the T7 capsid protein. The display vectors are useful for high copy number display or lower copy number display (with larger fusion). Compositions of the type described herein are useful in connection with methods for producing a virus displaying a protein or peptide of interest.

Use of a Phage-Display Method to Identify Peptides that Bind to a Tin Oxide Nanosheets.

PubMed

Nakazawa, Hikaru; Seta, Yasuko; Hirose, Tatsuya; Masuda, Yoshitake; Umetsu, Mitsuo

2018-01-01

Nanosheets of SnO2 which an n-type semiconductor with a rutile-type crystalline structure are predominantly used as gas sensors. SnO2 nanosheets have a tetragonal crystal structure where growth along the c-axis is suppressed to form a sheet. The major exposed facets of SnO2 nanosheets have {110}, {101} and {211} crystal planes along the a-axis, with the reduced {110} surface having a particularly high surface energy. Identifying peptides that bind to specific crystal planes by using peptide phage-display approach will increase the potential applications of metal oxide nanomaterials by fusing proteins with desirable active sites to peptides that adsorb at high density on the major exposed crystal plane of nanosheets. It may be possible to construct highly sensitive biosensors. The main objective of the present study is to identify peptides that adsorb preferentially to a SnO2 nanosheet by using peptide-phage display approach. Four milligrams of SnO2 nanosheet were mixed with 1011 plaque-forming units of Ph.D.-12 Phage Display Peptide Library. Phage-bound nanosheet particles were washed 10 times with 1 mL of phosphatebuffered saline containing 0.5% Tween 20. Phages bound to the nanosheet were eluted with three different buffers: (1) high-salt buffer containing 2 M NaCl (pH 7.5); (2) acidic buffer containing 200 mM Gly-HCl (pH 2.2); and (3) high-phosphate-ion buffer containing 500 mM NaH2PO4 (pH 7.5). The eluted phages were subjected to four or five rounds of biopanning. At each round, individual plaques were picked from the plates, and the amino acid sequences of the peptides were identified by DNA sequencing. The identified SnO2-binding peptides labeled with fluorescein isothiocyanate were synthesized. Adsorption isotherms were constructed at peptide concentrations ranging from 0.25 to 2.0 µM with 4mg of nanomaterials. We were determined the sequences of 11 clones with the high-salt buffer, 7 with the high-phosphateion buffers, and 6 with the acidic buffer and three peptides (SnO2BPn1, 2, and 3), two peptides (SnO2BPa1 and SnO2BPa2), and one peptide (SnO2BPp1) concentrated under each condition were selected respectively. All six selected peptides contained at least one histidine residue. In addition, the His-Asn-Leu (HNL) sequence was found in two of the peptides (SnO2BPa1 and SnO2BPa2). We constructed adsorption isotherms for the six selected peptides using 4mg of nanosheets. All six peptides were well adsorbed on the SnO2 nanosheet. The adsorption isotherms for SnO2 material with different structure revealed that SnO2BPn1, -2, and -3, and SnO2BPp1, preferentially bound to the spherical SnO2 nanoparticles. SnO2BPa2 preferentially bound to the SnO2 nanosheet, and SnO2BPa1 bound equally to both materials. This result suggested that SnO2BPa2 bound to a specific crystal plane of the nanosheet. The major exposed facet of the SnO2 crystal was the {110} plane, suggesting that SnO2BPa2 likely adsorbed on the {110} plane. SnO2BPn1, SnO2BPn2, SnO2BPn3, SnO2BPa1, and SnO2BPp1 also bound to the other metal oxides, in particular to ZrO2. At pH 7.5, peptides with a negative charge at pH 7.5 (pI 8.5-12) can bind to ZrO2 and SnO2, if the binding is mediated by electrostatic interactions. Thus, it is likely that these five peptides bind to metal oxides via electrostatic interactions. In contrast, SnO2BPa2 had a structurally specific affinity, binding more with the SnO2 nanosheet than with the spherical SnO2 nanoparticles or other metal oxides. We identified six peptides that adsorbed on a SnO2 nanosheet. Five of the selected peptides bound preferentially to spherical SnO2 nanoparticles rather than to the SnO2 nanosheet. Whereas, SnO2BPa2 exhibited specifically binding to the SnO2 nanosheet. Our results suggest that crystal plane recognition and material recognition by these peptides are mediated via different, independent mechanisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Cartographic symbol library considering symbol relations based on anti-aliasing graphic library

NASA Astrophysics Data System (ADS)

Mei, Yang; Li, Lin

2007-06-01

Cartographic visualization represents geographic information with a map form, which enables us retrieve useful geospatial information. In digital environment, cartographic symbol library is the base of cartographic visualization and is an essential component of Geographic Information System as well. Existing cartographic symbol libraries have two flaws. One is the display quality and the other one is relations adjusting. Statistic data presented in this paper indicate that the aliasing problem is a major factor on the symbol display quality on graphic display devices. So, effective graphic anti-aliasing methods based on a new anti-aliasing algorithm are presented and encapsulated in an anti-aliasing graphic library with the form of Component Object Model. Furthermore, cartographic visualization should represent feature relation in the way of correctly adjusting symbol relations besides displaying an individual feature. But current cartographic symbol libraries don't have this capability. This paper creates a cartographic symbol design model to implement symbol relations adjusting. Consequently the cartographic symbol library based on this design model can provide cartographic visualization with relations adjusting capability. The anti-aliasing graphic library and the cartographic symbol library are sampled and the results prove that the two libraries both have better efficiency and effect.

Characterization of particulate matter binding peptides screened from phage display.

PubMed

Liang Alvin, Aw Wei; Tanaka, Masayoshi; Okochi, Mina

2017-05-01

Particulate matter (PM), especially particulates with diameters of less than 2.5 μm, can penetrate the alveolar region and increase the risk of respiratory diseases. This has stimulated research efforts to develop detection methods so that counter measures can be taken. In this study, four PM binding peptides were obtained by phage display and binding characteristics of these peptides were investigated using the peptide array. The strongest binding peptide, WQDFGAVRSTRS, displayed a binding property, measured in terms of spot intensity, 11.4 times higher than that of the negative control, AAAAA. Inductively coupled plasma mass spectrometry (ICPMS) analysis of the transition metal compounds in the PM bound to the peptide spots was performed, and two peptides showed higher binding towards Cu and Zn compounds in PM. These results suggest that the screened peptides could serve as an indicator of transition metal compounds, which are related to adverse health effects, contained in PM. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials

NASA Astrophysics Data System (ADS)

Pappas, Charalampos G.; Shafi, Ramim; Sasselli, Ivan R.; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V.

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials.

PubMed

Pappas, Charalampos G; Shafi, Ramim; Sasselli, Ivan R; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy.

PubMed

Park, Seung-Hwan; Zheng, Jin Hai; Nguyen, Vu Hong; Jiang, Sheng-Nan; Kim, Dong-Yeon; Szardenings, Michael; Min, Jung Hyun; Hong, Yeongjin; Choy, Hyon E; Min, Jung-Joon

2016-01-01

Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvβ3, but weakly bound to αvβ3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvβ3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

Utilization of specific and non-specific peptide interactions with inorganic nanomaterials on the surface of bacteriophage M13: Methodologies towards phage supported bi-functional materials

NASA Astrophysics Data System (ADS)

Avery, Kendra Nicole

Many types of organisms create a variety of nano and micro scale materials from precursors available in their surrounding environments by a process called biomineralization. As scientists begin to understand how these organisms utilize specific and non-specific interactions with a variety of biopolymers such as chitin, peptides, proteins and nucleic acids with these precursors to create inorganic/organic composite materials, they have begun to wonder about the synthesis of other types of non-biologically templated synthetic techniques that might be possible. Bioengineered organisms and biopolymers have begun to be used for these types of studies. A variety of selection techniques exist for discovering biopolymers with an affinity for a target material, however, one of the most notable is a technique called peptide phage display. This is a technique that utilizes a commercially available randomized peptide library attached at the tip of the filamentous bacteriophage M13. In this dissertation capabilities of bacteriophage M13 are explored in regard to the creation of bi-functional nano materials by exploiting both specific peptide interactions as well as non-specific peptide interactions on the surface of the organism. Chapter 2 focuses on utilizing the specific peptide interactions of the randomized library at pIII in order to discover peptides with high binding affinity for a variety of nanomaterials. Selection studies called biopanning are performed on a variety of nanomaterials such as CaMoO4, allotropes of Ni, Fe2O3 and Fe3O4, and Rh and Pt with the fcc type crystal structure. Similarities and differences between peptides discovered for these materials are discussed. Chapter 3 focuses on utilizing the non-specific peptide interactions on the long axis of M13 called pVIII. The pVIII region consists of 2700 copies of the same 50 amino acid protein which as a negatively charged domain which is exposed to solution. The pVIII region therefore provides the surface of the phage with a negative charge on which nanomaterials can be supported. Metal salt precursors reduced in the presence of WT M13 are studied in this chapter. Metal salt precursors of Fe, Co, Ru, Rh and Pd seem to be the most effective at coating the surface of the phage based on the positively charged metal-aquo complexes formed in water, which are attracted to the negative pVIII region. Other types of reactions are explored with WT phage as a scaffold such as conversion chemistry in a polyol solvent to access several intermetallic phases as well as co-precipitation reactions to access ternary oxides. Chapter 4 focuses on combining research from chapter 2 and chapter 3 to create a bi-functional material that utilizes both specific and non-specific peptide interactions with inorganic materials on the surface of M13 to attach two different types of nanomaterials. The example provided here is a magnetically recoverable hydrogenation catalyst made up of a pVIII region coated with rhodium nanoparticles held in place by non-specific peptide interactions and a pIII region attached to iron oxide nanoparticles via specific peptide interactions. This is the first example in the literature of a commercially available pIII bioengineered M13 bacteriophage forming a bi-functional material. This research provides a methodology to design and build single and multi-component materials on the surface of bacteriophage M13 without the necessity for additional bioengineering and library characterization. The simplicity of use will make the technique available to a wider variety of researchers in the materials science community.

Mapping of binding epitopes of a human decay-accelerating factor monoclonal antibody capable of enhancing rituximab-mediated complement-dependent cytotoxicity.

PubMed

Guo, Bo; Ma, Zheng-wei; Li, Hua; Xu, Gui-lian; Zheng, Ping; Zhu, Bo; Wu, Yu-Zhang; Zou, Qiang

2008-08-01

Complement-dependent cytotoxicity (CDC) is thought to be one of the most important mechanisms of action of therapeutic monoclonal antibodies (mAbs). The decay-accelerating factor (DAF) overexpressed in certain tumors limits the CDC effect of the therapeutic anticancer antibodies. The use of DAF blocking antibodies targeted specifically at cancer cells in combination with immunotherapeutic mAbs of cancer may improve the therapeutic effect in cancer patients. In this study, the lysis of Raji cells mediated by CDC was determined after blocking DAF function by anti-DAF polyclonal antibody and 3 mAbs (DG3, DG9, DA11) prepared in our laboratory, respectively, in the presence of the anti-CD20 chimeric mAb rituximab. The binding domains of the three anti-DAF mAbs were identified using yeast surface display technique, and the mimic epitopes of mAb DG3 were screened from a random phage-display nonapeptide library. The results showed that blocking DAF function by anti-DAF polyclonal antibody enhanced complement-mediated killing of Raji cells. Among the 3 mAbs against DAF, only DG3 was found to be able to remarkably enhance the CDC effect of the therapeutic mAb rituximab. DG3 bound to the third short consensus repeat (SCR) of DAF. Binding of DG3 to immobilized DAF was inhibited by mimic epitope peptides screened from the peptide library. Our results suggest that a higher level of DAF expressed by certain tumor cells is significant to abolish the CDC effect of therapeutic anticancer antibodies, and mAbs binding to SCR3 can enhance the complement-mediated killing of Raji cells. It is of significance to identify the DAF epitopes required in inhibiting CDC not only for better understanding of the relationship between the structure and function of DAF, but also for designing and developing anti-DAF mAbs capable of enhancing CDC.

Computational and biophysical approaches to protein-protein interaction inhibition of Plasmodium falciparum AMA1/RON2 complex

NASA Astrophysics Data System (ADS)

Pihan, Emilie; Delgadillo, Roberto F.; Tonkin, Michelle L.; Pugnière, Martine; Lebrun, Maryse; Boulanger, Martin J.; Douguet, Dominique

2015-06-01

Invasion of the red blood cell by Plasmodium falciparum parasites requires formation of an electron dense circumferential ring called the Moving Junction (MJ). The MJ is anchored by a high affinity complex of two parasite proteins: Apical Membrane Antigen 1 ( PfAMA1) displayed on the surface of the parasite and Rhoptry Neck Protein 2 that is discharged from the parasite and imbedded in the membrane of the host cell. Structural studies of PfAMA1 revealed a conserved hydrophobic groove localized to the apical surface that coordinates RON2 and invasion inhibitory peptides. In the present work, we employed computational and biophysical methods to identify competitive P. falciparum AMA1-RON2 inhibitors with the goal of exploring the `druggability' of this attractive antimalarial target. A virtual screen followed by molecular docking with the PfAMA1 crystal structure was performed using an eight million compound collection that included commercial molecules, the ChEMBL malaria library and approved drugs. The consensus approach resulted in the selection of inhibitor candidates. We also developed a fluorescence anisotropy assay using a modified inhibitory peptide to experimentally validate the ability of the selected compounds to inhibit the AMA1-RON2 interaction. Among those, we identified one compound that displayed significant inhibition. This study offers interesting clues to improve the throughput and reliability of screening for new drug leads.

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae).

PubMed

Baek, Ji Hyeong; Lee, Si Hyeock

2010-06-01

To search for novel transcripts encoding biologically active venom components, a subtractive cDNA library specific to the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (ESTs) were clustered and assembled into 102 contigs (31 multiple sequences and 71 singletons). In total, 37 cDNAs were found in the library via BLASTx searching and manual annotation. Eight contigs (337 ESTs) encoding short venom peptides (10 to 16 amino acids) occupied 62% of the library. The deduced amino acid sequence (78 amino acids) of a novel venom peptide transcript shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known to be K(+) channel blockers, implying that this novel peptide may play a role in the paralysis of prey. In addition to phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, several transcripts encoding enzymes, including three metallopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, amines, and neurotransmitters, were also isolated from the library. The presence of a transcript encoding a putative insulin/insulin-like peptide binding protein suggests that solitary hunting wasps use their venom to control their prey, leading to larval growth cessation. The abundance of these venom components in the venom gland/sac and in the alimentary canal was confirmed by quantitative real-time PCR. Discovery of venom gland/sac-specific transcripts should promote further studies on biologically active components in the venom of solitary hunting wasps. Copyright 2010 Elsevier Ltd. All rights reserved.

Recent Advances Towards The Discovery Of Drug-Like Peptides De Novo

NASA Astrophysics Data System (ADS)

Goldflam, Michael; Ullman, Christopher

2015-12-01

Peptides are important natural molecules that possess functions as diverse as antibiotics, toxins, venoms and hormones, for example. However, whilst these peptides have useful properties, there are many targets and pathways that are not addressed through the activities of natural peptidic compounds. In these circumstances, directed evolution techniques, such as phage display, have been developed to sample the diverse chemical and structural repertoire of small peptides for useful means. In this review, we consider recent concepts that relate peptide structure to drug-like attributes and how these are incorporated within display technologies to deliver peptides de novo with valuable pharmaceutical properties.

Advance in phage display technology for bioanalysis.

PubMed

Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

2016-06-01

Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phage displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry

USDA-ARS?s Scientific Manuscript database

Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by po...

Exploitation of rolling circle amplification for the construction of large phage-display antibody libraries.

PubMed

Shahsavarian, Melody A; Le Minoux, Damien; Matti, Kalyankumar M; Kaveri, Srini; Lacroix-Desmazes, Sébastien; Boquet, Didier; Friboulet, Alain; Avalle, Bérangère; Padiolleau-Lefèvre, Séverine

2014-05-01

Phage display antibody libraries have proven to have a significant role in the discovery of therapeutic antibodies and polypeptides with desired biological and physicochemical properties. Obtaining a large and diverse phage display antibody library, however, is always a challenging task. Various steps of this technique can still undergo optimization in order to obtain an efficient library. In the construction of a single chain fragment variable (scFv) phage display library, the cloning of the scFv fragments into a phagemid vector is of crucial importance. An efficient restriction enzyme digestion of the scFv DNA leads to its proper ligation with the phagemid followed by its successful cloning and expression. Here, we are reporting a different approach to enhance the efficiency of the restriction enzyme digestion step. We have exploited rolling circle amplification (RCA) to produce a long strand of DNA with tandem repeats of scFv sequences, which is found to be highly susceptible to restriction digestion. With this important modification, we are able to construct a large phage display antibody library of naive SJL/J mice. The size of the library is estimated as ~10(8) clones. The number of clones containing a scFv fragment is estimated at 90%. Hence, the present results could considerably aid the utilization of the phage-display technique in order to get an efficiently large antibody library. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of Mycobacterium tuberculosis based on H37R(v) binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis.

PubMed

Yang, Hua; Qin, Lianhua; Wang, Yilong; Zhang, Bingbo; Liu, Zhonghua; Ma, Hui; Lu, Junmei; Huang, Xiaochen; Shi, Donglu; Hu, Zhongyi

2015-01-01

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl-Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 10(3) colony forming units (CFU)/mL H37R(v) was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 10(3) CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples.

Detection of Mycobacterium tuberculosis based on H37Rv binding peptides using surface functionalized magnetic microspheres coupled with quantum dots – a nano detection method for Mycobacterium tuberculosis

PubMed Central

Yang, Hua; Qin, Lianhua; Wang, Yilong; Zhang, Bingbo; Liu, Zhonghua; Ma, Hui; Lu, Junmei; Huang, Xiaochen; Shi, Donglu; Hu, Zhongyi

2015-01-01

Despite suffering from the major disadvantage of low sensitivity, microscopy of direct smear with the Ziehl–Neelsen stain is still broadly used for detection of acid-fast bacilli and diagnosis of tuberculosis. Here, we present a unique detection method of Mycobacterium tuberculosis (MTB) using surface functionalized magnetic microspheres (MMSs) coupled with quantum dots (QDs), conjugated with various antibodies and phage display-derived peptides. The principle is based upon the conformation of the sandwich complex composed of bacterial cells, MMSs, and QDs. The complex system is tagged with QDs for providing the fluorescent signal as part of the detection while magnetic separation is achieved by MMSs. The peptide ligand H8 derived from the phage display library Ph.D.-7 is developed for MTB cells. Using the combinations of MMS-polyclonal antibody+QD-H8 and MMS-H8+QD-H8, a strong signal of 103 colony forming units (CFU)/mL H37Rv was obtained with improved specificity. MS-H8+QD-H8 combination was further optimized by adjusting the concentrations of MMSs, QDs, and incubation time for the maximum detection signal. The limit of detection for MTB was found to reach 103 CFU/mL even for the sputum matrices. Positive sputum samples could be distinguished from control. Thus, this novel method is shown to improve the detection limit and specificity of MTB from the sputum samples, and to reduce the testing time for accurate diagnosis of tuberculosis, which needs further confirmation of more clinical samples. PMID:25565805

Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Martínez-Ceron, María C; Giudicessi, Silvana L; Marani, Mariela M; Albericio, Fernando; Cascone, Osvaldo; Erra-Balsells, Rosa; Camperi, Silvia A

2010-05-15

Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H(2)O), improving matrix crystallization. Peptide-bead cleavage with NH(4)OH was cheaper and safer than, yet as efficient as, NH(3)/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix. Copyright 2010 Elsevier Inc. All rights reserved.

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

PubMed

Das, Samir; Nag, Arundhati; Liang, JingXin; Bunck, David N; Umeda, Aiko; Farrow, Blake; Coppock, Matthew B; Sarkes, Deborah A; Finch, Amethist S; Agnew, Heather D; Pitram, Suresh; Lai, Bert; Yu, Mary Beth; Museth, A Katrine; Deyle, Kaycie M; Lepe, Bianca; Rodriguez-Rivera, Frances P; McCarthy, Amy; Alvarez-Villalonga, Belen; Chen, Ann; Heath, John; Stratis-Cullum, Dimitra N; Heath, James R

2015-11-02

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transformation of Human Cathelicidin LL-37 into Selective, Stable, and Potent Antimicrobial Compounds

PubMed Central

2015-01-01

This Letter reports a family of novel antimicrobial compounds obtained by combining peptide library screening with structure-based design. Library screening led to the identification of a human LL-37 peptide resistant to chymotrypsin. This d-amino-acid-containing peptide template was active against Escherichia coli but not methicillin-resistant Staphylococcus aureus (MRSA). It possesses a unique nonclassic amphipathic structure with hydrophobic defects. By repairing the hydrophobic defects, the peptide (17BIPHE2) gained activity against the ESKAPE pathogens, including Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species. In vitro, 17BIPHE2 could disrupt bacterial membranes and bind to DNA. In vivo, the peptide prevented staphylococcal biofilm formation in a mouse model of catheter-associated infection. Meanwhile, it boosted the innate immune response to further combat the infection. Because these peptides are potent, cell-selective, and stable to several proteases, they may be utilized to combat one or more ESKAPE pathogens. PMID:25061850

Screening of Pro-Asp Sequences Exposed on Bacteriophage M13 as an Ideal Anchor for Gold Nanocubes.

PubMed

Lee, Hwa Kyoung; Lee, Yujean; Kim, Hyori; Lee, Hye-Eun; Chang, Hyejin; Nam, Ki Tae; Jeong, Dae Hong; Chung, Junho

2017-09-15

Bacteriophages are thought to be ideal vehicles for linking antibodies to nanoparticles. Here, we define the sequence of peptides exposed as a fusion protein on M13 bacteriophages to yield optimal binding of gold nanocubes and efficient bacteriophage amplification. We generated five helper bacteriophage libraries using AE(X) 2 DP, AE(X) 3 DP, AE(X) 4 DP, AE(X) 5 DP, and AE(X) 6 DP as the exposed portion of pVIII, in which X was a randomized amino acid residue encoded by the nucleotide sequence NNK. Efficient phage amplification was achievable only in the AE(X) 2 DP, AE(X) 3 DP, and AE(X) 4 DP libraries. Through biopanning with gold nanocubes, we enriched the phage clones and selected the clone with the highest fold change after enrichment. This clone displayed Pro-Asp on the surface of the bacteriophage and had amplification yields similar to those of the wild-type helper bacteriophage (VCSM13). The clone displayed even binding of gold nanocubes along its length and minimal aggregation after binding. We conclude that, for efficient amplification, the exposed pVIII amino acid length should be limited to six residues and Ala-Glu-Pro-Asp-Asp-Pro (AEPDDP) is the ideal fusion protein sequence for guaranteeing the optimal formation of a complex with gold nanocubes.

Inhibition of Herpes Simplex Virus gD and Lymphotoxin-α Binding to HveA by Peptide Antagonists

PubMed Central

Sarrias, Maria Rosa; Whitbeck, J. Charles; Rooney, Isabelle; Spruce, Lynn; Kay, Brian K.; Montgomery, Rebecca I.; Spear, Patricia G.; Ware, Carl F.; Eisenberg, Roselyn J.; Cohen, Gary H.; Lambris, John D.

1999-01-01

The herpesvirus entry mediator A (HveA) is a recently characterized member of the tumor necrosis factor receptor family that mediates the entry of most herpes simplex virus type 1 (HSV-1) strains into mammalian cells. Studies on the interaction of HSV-1 with HveA have shown that of all the viral proteins involved in uptake, only gD has been shown to bind directly to HveA, and this binding mediates viral entry into cells. In addition to gD binding to HveA, the latter has been shown to interact with proteins of tumor necrosis factor receptor-associated factor family, lymphotoxin-α (LT-α), and a membrane-associated protein referred to as LIGHT. To study the relationship between HveA, its natural ligands, and the viral proteins involved in HSV entry into cells, we have screened two phage-displayed combinatorial peptide libraries for peptide ligands of a recombinant form of HveA. Affinity selection experiments yielded two peptide ligands, BP-1 and BP-2, which could block the interaction between gD and HveA. Of the two peptides, only BP-2 inhibited HSV entry into CHO cells transfected with an HveA-expressing plasmid. When we analyzed these peptides for the ability to interfere with HveA binding to its natural ligand LT-α, we found that BP-1 inhibited the interaction of cellular LT-α with HveA. Thus, we have dissected the sites of interaction between the cell receptor, its natural ligand LT-α and gD, the virus-specific protein involved in HSV entry into cells. PMID:10364318

Design and screening of M13 phage display cDNA libraries.

PubMed

Georgieva, Yuliya; Konthur, Zoltán

2011-02-17

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.

Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts.

PubMed

Bąchor, Remigiusz; Cydzik, Marzena; Rudowska, Magdalena; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew

2012-08-01

A rapid and straightforward method for high-throughput analysis of single resin beads from one-bead-one-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ɛ-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry.

The Effect of Face-Front Display on the Circulation of Books in a Public Library.

ERIC Educational Resources Information Center

Long, Sarah P.

This study considers the theories of impulse buying in an examination of the effects on circulation of library books when books are displayed face front (with all or most of the book jacket showing) as opposed to spine front. Reviews of the literature on consumer behavior and on library displays support the hypothesis of this study, i.e., that…

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Entomopathogenic bacteria use multiple mechanisms for bioactive peptide library design

NASA Astrophysics Data System (ADS)

Cai, Xiaofeng; Nowak, Sarah; Wesche, Frank; Bischoff, Iris; Kaiser, Marcel; Fürst, Robert; Bode, Helge. B.

2017-04-01

The production of natural product compound libraries has been observed in nature for different organisms such as bacteria, fungi and plants; however, little is known about the mechanisms generating such chemically diverse libraries. Here we report mechanisms leading to the biosynthesis of the chemically diverse rhabdopeptide/xenortide peptides (RXPs). They are exclusively present in entomopathogenic bacteria of the genera Photorhabdus and Xenorhabdus that live in symbiosis with nematodes delivering them to insect prey, which is killed and utilized for nutrition by both nematodes and bacteria. Chemical diversity of the biologically active RXPs results from a combination of iterative and flexible use of monomodular nonribosomal peptide synthetases including substrate promiscuity, enzyme cross-talk and enzyme stoichiometry as shown by in vivo and in vitro experiments. Together, this highlights several of nature's methods for diversification, or evolution, of natural products and sheds light on the biosynthesis of the bioactive RXPs.

Genetically engineered peptides for inorganics: study of an unconstrained bacterial display technology and bulk aluminum alloy.

PubMed

Adams, Bryn L; Finch, Amethist S; Hurley, Margaret M; Sarkes, Deborah A; Stratis-Cullum, Dimitra N

2013-09-06

The first-ever peptide biomaterial discovery using an unconstrained engineered bacterial display technology is reported. Using this approach, we have developed genetically engineered peptide binders for a bulk aluminum alloy and use molecular dynamics simulation of peptide conformational fluctuations to demonstrate sequence-dependent, structure-function relationships for metal and metal oxide interactions. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A RHAMM mimetic peptide blocks hyaluronan signaling and reduces inflammation and fibrogenesis in excisional skin wounds.

PubMed

Tolg, Cornelia; Hamilton, Sara R; Zalinska, Ewa; McCulloch, Lori; Amin, Ripal; Akentieva, Natalia; Winnik, Francoise; Savani, Rashmin; Bagli, Darius J; Luyt, Len G; Cowman, Mary K; McCarthy, Jim B; Turley, Eva A

2012-10-01

Hyaluronan is activated by fragmentation and controls inflammation and fibroplasia during wound repair and diseases (eg, cancer). Hyaluronan-binding peptides were identified that modify fibrogenesis during skin wound repair. Peptides were selected from 7- to 15mer phage display libraries by panning with hyaluronan-Sepharose beads and assayed for their ability to block fibroblast migration in response to hyaluronan oligosaccharides (10 kDa). A 15mer peptide (P15-1), with homology to receptor for hyaluronan mediated motility (RHAMM) hyaluronan binding sequences, was the most effective inhibitor. P15-1 bound to 10-kDa hyaluronan with an affinity of K(d) = 10(-7) and appeared to specifically mimic RHAMM since it significantly reduced binding of hyaluronan oligosaccharides to recombinant RHAMM but not to recombinant CD44 or TLR2,4, and altered wound repair in wild-type but not RHAMM(-/-) mice. One topical application of P15-1 to full-thickness excisional rat wounds significantly reduced wound macrophage number, fibroblast number, and blood vessel density compared to scrambled, negative control peptides. Wound collagen 1, transforming growth factor β-1, and α-smooth muscle actin were reduced, whereas tenascin C was increased, suggesting that P15-1 promoted a form of scarless healing. Signaling/microarray analyses showed that P15-1 blocks RHAMM-regulated focal adhesion kinase pathways in fibroblasts. These results identify a new class of reagents that attenuate proinflammatory, fibrotic repair by blocking hyaluronan oligosaccharide signaling. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Comparative Analysis of the Antimicrobial Activities of Plant Defensin-Like and Ultrashort Peptides against Food-Spoiling Bacteria.

PubMed

Kraszewska, Joanna; Beckett, Michael C; James, Tharappel C; Bond, Ursula

2016-07-15

Antimicrobial peptides offer potential as novel therapeutics to combat food spoilage and poisoning caused by pathogenic and nonpathogenic bacteria. Our previous studies identified the peptide human beta-defensin 3 (HBD3) as a potent antimicrobial agent against a wide range of beer-spoiling bacteria. Thus, HBD3 is an excellent candidate for development as an additive to prevent food and beverage spoilage. To expand the repertoire of peptides with antimicrobial activity against bacteria associated with food spoilage and/or food poisoning, we carried out an in silico discovery pipeline to identify peptides with structure and activity similar to those of HBD3, focusing on peptides of plant origin. Using a standardized assay, we compared the antimicrobial activities of nine defensin-like plant peptides to the activity of HBD3. Only two of the peptides, fabatin-2 and Cp-thionin-2, displayed antimicrobial activity; however, the peptides differed from HBD3 in being sensitive to salt and were thermostable. We also compared the activities of several ultrashort peptides to that of HBD3. One of the peptides, the synthetic tetrapeptide O3TR, displayed biphasic antimicrobial activity but had a narrower host range than HBD3. Finally, to determine if the peptides might act in concert to improve antimicrobial activity, we compared the activities of the peptides in pairwise combinations. The plant defensin-like peptides fabatin-2 and Cp-thionin-2 displayed a synergistic effect with HBD3, while O3TR was antagonistic. Thus, some plant defensin-like peptides are effective antimicrobials and may act in concert with HBD3 to control bacteria associated with food spoilage and food poisoning. Food spoilage and food poisoning caused by bacteria can have major health and economic implications for human society. With the rise in resistance to conventional antibiotics, there is a need to identify new antimicrobials to combat these outbreaks in our food supply. Here we screened plant peptide databases to identify peptides that share structural similarity with the human defensin peptide HBD3, which has known antimicrobial activity against food-spoiling bacteria. We show that two of the plant peptides display antimicrobial activity against bacteria associated with food spoilage. When combined with HBD3, the peptides are highly effective. We also analyzed the activity of an easily made ultrashort synthetic peptide, O3TR. We show that this small peptide also displays antimicrobial activity against food-spoiling bacteria but is not as effective as HBD3 or the plant peptides. The plant peptides identified are good candidates for development as natural additives to prevent food spoilage. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Targeting neuropilin-1 in human leukemia and lymphoma.

PubMed

Karjalainen, Katja; Jaalouk, Diana E; Bueso-Ramos, Carlos E; Zurita, Amado J; Kuniyasu, Akihiko; Eckhardt, Bedrich L; Marini, Frank C; Lichtiger, Benjamin; O'Brien, Susan; Kantarjian, Hagop M; Cortes, Jorge E; Koivunen, Erkki; Arap, Wadih; Pasqualini, Renata

2011-01-20

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/Tyr-Any-Leu-Arg-Ser (F(F)/(Y)XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence (D)(KLAKLAK)₂. Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for ligand-directed therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-(D)(KLAKLAK)₂ is a promising drug candidate in this setting.

Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting.

PubMed

Staquicini, Daniela I; Rangel, Roberto; Guzman-Rojas, Liliana; Staquicini, Fernanda I; Dobroff, Andrey S; Tarleton, Christy A; Ozbun, Michelle A; Kolonin, Mikhail G; Gelovani, Juri G; Marchiò, Serena; Sidman, Richard L; Hajjar, Katherine A; Arap, Wadih; Pasqualini, Renata

2017-06-26

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

One-Bead-Two-Compound Thioether Bridged Macrocyclic γ-AApeptide Screening Library against EphA2.

PubMed

Shi, Yan; Challa, Sridevi; Sang, Peng; She, Fengyu; Li, Chunpu; Gray, Geoffrey M; Nimmagadda, Alekhya; Teng, Peng; Odom, Timothy; Wang, Yan; van der Vaart, Arjan; Li, Qi; Cai, Jianfeng

2017-11-22

Identification of molecular ligands that recognize peptides or proteins is significant but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce, and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented one-bead-two-compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound that tightly bound to EphA2 (K d = 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform for biomacromolecular surface recognition and function modulation.

Stress-Driven Selection of Novel Phenotypes

NASA Technical Reports Server (NTRS)

Fox, George E.; Stepaov, Victor G.; Liu, Yamei

2011-01-01

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

PubMed

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

The structure of a thermophilic kinase shapes fitness upon random circular permutation

PubMed Central

Jones, Alicia M.; Mehta, Manan M.; Thomas, Emily E.; Atkinson, Joshua T.; Segall-Shapiro, Thomas H.; Liu, Shirley; Silberg, Jonathan J.

2016-01-01

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement where native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein’s functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AK with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and they reveal a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection. PMID:26976658

The Structure of a Thermophilic Kinase Shapes Fitness upon Random Circular Permutation.

PubMed

Jones, Alicia M; Mehta, Manan M; Thomas, Emily E; Atkinson, Joshua T; Segall-Shapiro, Thomas H; Liu, Shirley; Silberg, Jonathan J

2016-05-20

Proteins can be engineered for synthetic biology through circular permutation, a sequence rearrangement in which native protein termini become linked and new termini are created elsewhere through backbone fission. However, it remains challenging to anticipate a protein's functional tolerance to circular permutation. Here, we describe new transposons for creating libraries of randomly circularly permuted proteins that minimize peptide additions at their termini, and we use transposase mutagenesis to study the tolerance of a thermophilic adenylate kinase (AK) to circular permutation. We find that libraries expressing permuted AKs with either short or long peptides amended to their N-terminus yield distinct sets of active variants and present evidence that this trend arises because permuted protein expression varies across libraries. Mapping all sites that tolerate backbone cleavage onto AK structure reveals that the largest contiguous regions of sequence that lack cleavage sites are proximal to the phosphotransfer site. A comparison of our results with a range of structure-derived parameters further showed that retention of function correlates to the strongest extent with the distance to the phosphotransfer site, amino acid variability in an AK family sequence alignment, and residue-level deviations in superimposed AK structures. Our work illustrates how permuted protein libraries can be created with minimal peptide additions using transposase mutagenesis, and it reveals a challenge of maintaining consistent expression across permuted variants in a library that minimizes peptide additions. Furthermore, these findings provide a basis for interpreting responses of thermophilic phosphotransferases to circular permutation by calibrating how different structure-derived parameters relate to retention of function in a cellular selection.

Control of Surface Chemistry, Substrate Stiffness, and Cell Function in a Novel Terpolymer Methacrylate Library

PubMed Central

Joy, Abraham; Cohen, Daniel M.; Luk, Arnold; Anim-Danso, Emmanuel; Chen, Christopher; Kohn, Joachim

2011-01-01

A focused library of methacrylate terpolymers was synthesized to explore the effects of varying surface chemistry and adhesive peptide ligands on cell function. The chemical diversity of methacrylate monomers enabled construction of a library of polymers in which one can systematically vary the chemical composition to achieve a wide range of contact angle, Young's modulus, and Tg values. Furthermore, the materials were designed to allow surface immobilization of bioactive peptides. We then examined the effects of these material compositions on protein adsorption and cell attachment, proliferation, and differentiation. We observed that chemical composition of the polymers was an important determinant for NIH 3T3 cell attachment and proliferation, as well as human mesenchymal stem cell differentiation, and correlated directly with the ability of the polymers to adsorb proteins that mediate cell adhesion. Importantly, functionalization of the methacrylate terpolymer library with an adhesive GRGDS peptide normalized cellular responses. RGD-functionalized polymers uniformly exhibited robust attachment, proliferation, and differentiation irrespective of the underlying substrate chemistry. These studies provide a library-based approach to rapidly explore the biological functionality of biomaterials with a wide range of compositions, and highlights the importance of cell and protein cell adhesion in predicting their performance. PMID:21226505

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Hua; Jiang Lifang; Fang Danyun

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosenmore » for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.« less

Flunarizine Prevents Hepatitis C Virus Membrane Fusion in a Genotype-dependent Manner by Targeting the Potential Fusion Peptide within E1

PubMed Central

Perin, Paula M.; Haid, Sibylle; Brown, Richard J. P.; Doerrbecker, Juliane; Schulze, Kai; Zeilinger, Carsten; von Schaewen, Markus; Heller, Brigitte; Vercauteren, Koen; Luxenburger, Eva; Baktash, Yasmine M.; Vondran, Florian W. R.; Speerstra, Sietkse; Awadh, Abdullah; Mukhtarov, Furkat; Schang, Luis M; Kirschning, Andreas; Müller, Rolf; Guzman, Carlos A.; Kaderali, Lars; Randall, Glenn; Meuleman, Philip; Ploss, Alexander; Pietschmann, Thomas

2015-01-01

To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies. PMID:26248546

A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Sijun, E-mail: sliu@iastate.ed; Sivakumar, S., E-mail: sivaento@iastate.ed; Sparks, Wendy O., E-mail: wosparks@iastate.ed

2010-05-25

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptakemore » of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.« less

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

PubMed Central

Hall Sedlak, Ruth; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan

2012-01-01

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo. PMID:22286990

Development of Bacterial Display Peptides for use in Biosensing Applications

DTIC Science & Technology

2012-09-01

performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB...reagent, affinity reagent, bacterial display, multi-scale modeling, docking, protective antigen , SEB, biosensing 16. SECURITY CLASSIFICATION OF: 17...performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be

Antimicrobial Lactoferrin Peptides: The Hidden Players in the Protective Function of a Multifunctional Protein

PubMed Central

Sinha, Mau; Kaushik, Sanket; Kaur, Punit; Singh, Tej P.

2013-01-01

Lactoferrin is a multifunctional, iron-binding glycoprotein which displays a wide array of modes of action to execute its primary antimicrobial function. It contains various antimicrobial peptides which are released upon its hydrolysis by proteases. These peptides display a similarity with the antimicrobial cationic peptides found in nature. In the current scenario of increasing resistance to antibiotics, there is a need for the discovery of novel antimicrobial drugs. In this context, the structural and functional perspectives on some of the antimicrobial peptides found in N-lobe of lactoferrin have been reviewed. This paper provides the comparison of lactoferrin peptides with other antimicrobial peptides found in nature as well as interspecies comparison of the structural properties of these peptides within the native lactoferrin. PMID:23554820

Short, multiple-stranded β-hairpin peptides have antimicrobial potency with high selectivity and salt resistance.

PubMed

Chou, Shuli; Shao, Changxuan; Wang, Jiajun; Shan, Anshan; Xu, Lin; Dong, Na; Li, Zhongyu

2016-01-01

The β-hairpin structure has been proposed to exhibit potent antimicrobial properties with low cytotoxicity, thus, multiple β-hairpin structures have been proved to be highly stable in structures containing tightly packed hydrophobic cores. The aim of this study was to develop peptide-based synthetic strategies for generating short, but effective AMPs as inexpensive antimicrobial agents. Multiple-stranded β-hairpin peptides with the same β-hairpin unit, (WRXxRW)n where n=1, 2, 3, or 4 and Xx represent the turn sequence, were synthesized, and their potential as antimicrobial agents was evaluated. Owning to the tightly packed hydrophobic core and paired Trp of this multiple-stranded β-hairpin structure, all the 12-residues peptides exhibited high cell selectivity towards bacterial cells over human red blood cells (hRBCs), and the peptide W2 exhibited stronger antimicrobial activities with the MIC values of 2-8μM against various tested bacteria. Not only that, but W2 also showed obvious synergy with streptomycin and chloramphenicol against Escherichia coli, and displayed synergy with ciprofloxacin against Staphylococcus aureus with the FICI values ⩽0.5. Fluorescence spectroscopy and electron microscopy analyses indicated that W2 kills microbial cells by permeabilizing the cell membrane and damaging membrane integrity. Collectively, based on the multiple β-hairpin peptides, the ability to develop libraries of short and effective peptides will be a powerful approach to the discovery of novel antimicrobial agents. We successfully screened a peptide W2 ((WRPGRW)2) from a series of multiple-stranded β-hairpin antimicrobial peptides based on the "S-shaped" motif that induced the formation of a globular structure, and Trp zipper was used to replace the disulfide bonds to reduce the cost of production. This novel structure applied to AMPs improved cell selectivity and salt stability. The findings of this study will promote the development of peptide-based antimicrobial biomaterials. Further exploration of these AMPs will allow for diverse biotechnological and clinical applications such as biomedical coating, food storaging, and animal feeding. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib

PubMed Central

Mathea, Sebastian; Abdul Azeez, Kamal R.; Salah, Eidarus; Tallant, Cynthia; Wolfreys, Finn; Konietzny, Rebecca; Fischer, Roman; Lou, Hua Jane; Brennan, Paul E.; Schnapp, Gisela; Pautsch, Alexander; Kessler, Benedikt M.; Turk, Benjamin E.; Knapp, Stefan

2017-01-01

The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The co-crystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions −1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs. PMID:26999302

Epitope mapping: the first step in developing epitope-based vaccines.

PubMed

Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael

2007-01-01

Antibodies are an effective line of defense in preventing infectious diseases. Highly potent neutralizing antibodies can intercept a virus before it attaches to its target cell and, thus, inactivate it. This ability is based on the antibodies' specific recognition of epitopes, the sites of the antigen to which antibodies bind. Thus, understanding the antibody/epitope interaction provides a basis for the rational design of preventive vaccines. It is assumed that immunization with the precise epitope, corresponding to an effective neutralizing antibody, would elicit the generation of similarly potent antibodies in the vaccinee. Such a vaccine would be a 'B-cell epitope-based vaccine', the implementation of which requires the ability to backtrack from a desired antibody to its corresponding epitope. In this article we discuss a range of methods that enable epitope discovery based on a specific antibody. Such a reversed immunological approach is the first step in the rational design of an epitope-based vaccine. Undoubtedly, the gold standard for epitope definition is x-ray analyses of crystals of antigen:antibody complexes. This method provides atomic resolution of the epitope; however, it is not readily applicable to many antigens and antibodies, and requires a very high degree of sophistication and expertise. Most other methods rely on the ability to monitor the binding of the antibody to antigen fragments or mutated variations. In mutagenesis of the antigen, loss of binding due to point modification of an amino acid residue is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping are also useful. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have been developed, such as Mapitope, which has recently been found to be effective in mapping conformational discontinuous epitopes. The pros and cons of various approaches towards epitope mapping are also discussed.

Co-immobilization of adhesive peptides and VEGF within a dextran-based coating for vascular applications.

PubMed

Noel, Samantha; Fortier, Charles; Murschel, Frederic; Belzil, Antoine; Gaudet, Guillaume; Jolicoeur, Mario; De Crescenzo, Gregory

2016-06-01

Multifunctional constructs providing a proper environment for adhesion and growth of selected cell types are needed for most tissue engineering and regenerative medicine applications. In this context, vinylsulfone (VS)-modified dextran was proposed as a matrix featuring low-fouling properties as well as multiple versatile moieties. The displayed VS groups could indeed react with thiol, amine or hydroxyl groups, be it for surface grafting, crosslinking or subsequent tethering of biomolecules. In the present study, a library of dextran-VS was produced, grafted to aminated substrates and characterized in terms of degree of VS modification (%VS), cell-repelling properties and potential for the oriented grafting of cysteine-tagged peptides. As a bioactive coating of vascular implants, ECM peptides (e.g. RGD) as well as vascular endothelial growth factor (VEGF) were co-immobilized on one of the most suitable dextran-VS coating (%VS=ca. 50% of saccharides units). Both RGD and VEGF were efficiently tethered at high densities (ca. 1nmol/cm(2) and 50fmol/cm(2), respectively), and were able to promote endothelial cell adhesion as well as proliferation. The latter was enhanced to the same extent as with soluble VEGF and proved selective to endothelial cells over smooth muscle cells. Altogether, multiple biomolecules could be efficiently incorporated into a dextran-VS construct, while maintaining their respective biological activity. This work addresses the need for multifunctional coatings and selective cell response inherent to many tissue engineering and regenerative medicine applications, for instance, vascular graft. More specifically, a library of dextrans was first generated through vinylsulfone (VS) modification. Thoroughly selected dextran-VS provided an ideal platform for unbiased study of cell response to covalently grafted biomolecules. Considering that processes such as healing and angiogenesis require multiple factors acting synergistically, vascular endothelial growth factor (VEGF) was then co-immobilized with the cell adhesive RGD peptide within our dextran coating through a relevant strategy featuring orientation and specificity. Altogether, both adhesive and proliferative cues could be incorporated into our construct with additive, if not synergetic, effects. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity

PubMed Central

Koparde, Vishal N.; Jameson-Lee, Maximilian; Elnasseh, Abdelrhman G.; Scalora, Allison F.; Kobulnicky, David J.; Serrano, Myrna G.; Roberts, Catherine H.; Buck, Gregory A.; Neale, Michael C.; Nixon, Daniel E.; Toor, Amir A.

2017-01-01

Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD. PMID:28800601

Sequence homology between HLA-bound cytomegalovirus and human peptides: A potential trigger for alloreactivity.

PubMed

Hall, Charles E; Koparde, Vishal N; Jameson-Lee, Maximilian; Elnasseh, Abdelrhman G; Scalora, Allison F; Kobulnicky, David J; Serrano, Myrna G; Roberts, Catherine H; Buck, Gregory A; Neale, Michael C; Nixon, Daniel E; Toor, Amir A

2017-01-01

Human cytomegalovirus (hCMV) reactivation may often coincide with the development of graft-versus-host-disease (GVHD) in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n = 50; matched related donor (MRD), n = 27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (≥6) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50<500nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, an average of 29,658 matched the MRD DRP alloreactive peptides and 52,910 matched MUD DRP peptides (p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner. hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset. Analysis of patients with GVHD identified potential cross reactive peptide expression within affected organs. We propose that hCMV peptide sequence homology with human alloreactive peptides may contribute to the pathophysiology of GVHD.

Phage display of an intracellular carboxylesterase of Bacillus subtilis: comparison of Sec and Tat pathway export capabilities.

PubMed

Dröge, Melloney J; Boersma, Ykelien L; Braun, Peter G; Buining, Robbert Jan; Julsing, Mattijs K; Selles, Karin G A; van Dijl, Jan Maarten; Quax, Wim J

2006-07-01

Using the phage display technology, a protein can be displayed at the surface of bacteriophages as a fusion to one of the phage coat proteins. Here we describe development of this method for fusion of an intracellular carboxylesterase of Bacillus subtilis to the phage minor coat protein g3p. The carboxylesterase gene was cloned in the g3p-based phagemid pCANTAB 5E upstream of the sequence encoding phage g3p and downstream of a signal peptide-encoding sequence. The phage-bound carboxylesterase was correctly folded and fully enzymatically active, as determined from hydrolysis of the naproxen methyl ester with Km values of 0.15 mM and 0.22 mM for the soluble and phage-displayed carboxylesterases, respectively. The signal peptide directs the encoded fusion protein to the cell membrane of Escherichia coli, where phage particles are assembled. In this study, we assessed the effects of several signal peptides, both Sec dependent and Tat dependent, on the translocation of the carboxylesterase in order to optimize the phage display of this enzyme normally restricted to the cytoplasm. Functional display of Bacillus carboxylesterase NA could be achieved when Sec-dependent signal peptides were used. Although a Tat-dependent signal peptide could direct carboxylesterase translocation across the inner membrane of E. coli, proper assembly into phage particles did not seem to occur.

Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

PubMed

Rabinowitz, Joshua A; Lainson, John C; Johnston, Stephen Albert; Diehnelt, Chris W

2018-02-06

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

Genomic Approaches for Detection and Treatment of Breast Cancer

DTIC Science & Technology

2010-07-01

cloning them in phage display vectors. We are characterizing the libraries and trying to figure out how best to screen them. We ran into the problem...auto-antibody screening project onto glass slides for screening purposes. We have abandoned this aim in that we switched our approach to a phage ...display library which does not require glass slide. We made our first comprehensive library in a T7 display vector. Task 9 (Months 24-36) We will

Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

PubMed

Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

2017-07-01

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p < 0.01), while significantly increasing hBMSC adhesion strength (τ 50 , p < 0.01). MSCs demonstrated significantly greater adhesion strength to DPI-VTK compared to other cell types, while attachment of MC3T3 pre-osteoblasts and murine fibroblasts was limited (p < 0.01). MSCs on DPI-VTK coated surfaces also demonstrated increased spreading compared to pre-osteoblasts and fibroblasts. MSCs cultured on DPI-VTK coated apatite films exhibited significantly greater proliferation compared to controls (p < 0.001). Moreover, early and late stage osteogenic differentiation markers were elevated on DPI-VTK coated apatite films compared to controls. Taken together, phage display can identify non-obvious cell and material specific peptides to increase human MSC adhesion strength to specific biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysing the substrate multispecificity of a proton-coupled oligopeptide transporter using a dipeptide library

PubMed Central

Ito, Keisuke; Hikida, Aya; Kawai, Shun; Lan, Vu Thi Tuyet; Motoyama, Takayasu; Kitagawa, Sayuri; Yoshikawa, Yuko; Kato, Ryuji; Kawarasaki, Yasuaki

2013-01-01

Peptide uptake systems that involve members of the proton-coupled oligopeptide transporter (POT) family are conserved across all organisms. POT proteins have characteristic substrate multispecificity, with which one transporter can recognize as many as 8,400 types of di/tripeptides and certain peptide-like drugs. Here we characterize the substrate multispecificity of Ptr2p, a major peptide transporter of Saccharomyces cerevisiae, using a dipeptide library. The affinities (Ki) of di/tripeptides toward Ptr2p show a wide distribution range from 48 mM to 0.020 mM. This substrate multispecificity indicates that POT family members have an important role in the preferential uptake of vital amino acids. In addition, we successfully establish high performance ligand affinity prediction models (97% accuracy) using our comprehensive dipeptide screening data in conjunction with simple property indices for describing ligand molecules. Our results provide an important clue to the development of highly absorbable peptides and their derivatives including peptide-like drugs. PMID:24060756

Biologically-Inspired Peptide Reagents for Enhancing IMS-MS Analysis of Carbohydrates

NASA Astrophysics Data System (ADS)

Bohrer, Brian C.; Clemmer, David E.

2011-09-01

The binding properties of a peptidoglycan recognition protein are translated via combinatorial chemistry into short peptides. Non-adjacent histidine, tyrosine, and arginine residues in the protein's binding cleft that associate specifically with the glycan moiety of a peptidoglycan substrate are incorporated into linear sequences creating a library of 27 candidate tripeptide reagents (three possible residues permutated across three positions). Upon electrospraying the peptide library and carbohydrate mixtures, some noncovalent complexes are observed. The binding efficiencies of the peptides vary according to their amino acid composition as well as the disaccharide linkage and carbohydrate ring-type. In addition to providing a charge-carrier for the carbohydrate, peptide reagents can also be used to differentiate carbohydrate isomers by ion mobility spectrometry. The utility of these peptide reagents as a means of enhancing ion mobility analysis of carbohydrates is illustrated by examining four glucose-containing disaccharide isomers, including a pair that is not resolved by ion mobility alone. The specificity and stoichiometry of the peptide-carbohydrate complexes are also investigated. Trihistidine demonstrates both suitable binding efficiency and successful resolution of disaccharides isomers, suggesting it may be a useful reagent in IMS analyses of carbohydrates.

Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library.

PubMed

Song, Suk-yoon; Hur, Byung-ung; Lee, Kyung-woo; Choi, Hyo-jung; Kim, Sung-soo; Kang, Goo; Cha, Sang-hoon

2009-03-31

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 x 10(7) combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a 1.5 x 10(9) combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 x 10(7) heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule.

Stabilization of exosome-targeting peptides via engineered glycosylation.

PubMed

Hung, Michelle E; Leonard, Joshua N

2015-03-27

Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A computational docking study on the pH dependence of peptide binding to HLA-B27 sub-types differentially associated with ankylosing spondylitis

NASA Astrophysics Data System (ADS)

Serçinoğlu, Onur; Özcan, Gülin; Kabaş, Zeynep Kutlu; Ozbek, Pemra

2016-07-01

A single amino acid difference (Asp116His), having a key role in a pathogenesis pathway, distinguishes HLA-B*27:05 and HLA-B*27:09 sub-types as associated and non-associated with ankylosing spondylitis, respectively. In this study, molecular docking simulations were carried out with the aim of comprehending the differences in the binding behavior of both alleles at varying pH conditions. A library of modeled peptides was formed upon single point mutations aiming to address the effect of 20 naturally occurring amino acids at the binding core peptide positions. For both alleles, computational docking was applied using Autodock 4.2. Obtained free energies of binding (FEB) were compared within the peptide library and between the alleles at varying pH conditions. The amino acid preferences of each position were studied enlightening the role of each on binding. The preferred amino acids for each position of pVIPR were found to be harmonious with experimental studies. Our results indicate that, as the pH is lowered, the capacity of HLA-B*27:05 to bind peptides in the library is largely lost. Hydrogen bonding analysis suggests that the interaction between the main anchor positions of pVIPR and their respective binding pocket residues are affected from the pH the most, causing an overall shift in the FEB profiles.

A computational docking study on the pH dependence of peptide binding to HLA-B27 sub-types differentially associated with ankylosing spondylitis.

PubMed

Serçinoğlu, Onur; Özcan, Gülin; Kabaş, Zeynep Kutlu; Ozbek, Pemra

2016-07-01

A single amino acid difference (Asp116His), having a key role in a pathogenesis pathway, distinguishes HLA-B*27:05 and HLA-B*27:09 sub-types as associated and non-associated with ankylosing spondylitis, respectively. In this study, molecular docking simulations were carried out with the aim of comprehending the differences in the binding behavior of both alleles at varying pH conditions. A library of modeled peptides was formed upon single point mutations aiming to address the effect of 20 naturally occurring amino acids at the binding core peptide positions. For both alleles, computational docking was applied using Autodock 4.2. Obtained free energies of binding (FEB) were compared within the peptide library and between the alleles at varying pH conditions. The amino acid preferences of each position were studied enlightening the role of each on binding. The preferred amino acids for each position of pVIPR were found to be harmonious with experimental studies. Our results indicate that, as the pH is lowered, the capacity of HLA-B*27:05 to bind peptides in the library is largely lost. Hydrogen bonding analysis suggests that the interaction between the main anchor positions of pVIPR and their respective binding pocket residues are affected from the pH the most, causing an overall shift in the FEB profiles.

Identification of avocado (Persea americana) pulp proteins by nano-LC-MS/MS via combinatorial peptide ligand libraries.

PubMed

Esteve, Clara; D'Amato, Alfonsina; Marina, María Luisa; García, María Concepción; Righetti, Pier Giorgio

2012-09-01

Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse-phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low-abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone reductase like protein. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

PubMed

Mulder, Gwenn E; Quarles van Ufford, H Linda C; van Ameijde, Jeroen; Brouwer, Arwin J; Kruijtzer, John A W; Liskamp, Rob M J

2013-04-28

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

PubMed

Yeung, Yik A; Wittrup, K Dane

2002-01-01

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

Construction of human antibody gene libraries and selection of antibodies by phage display.

PubMed

Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

2014-01-01

Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

PubMed

Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

2009-09-01

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Principles and application of antibody libraries for infectious diseases.

PubMed

Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

2014-12-01

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

PubMed

Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

2015-03-23

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

Old Books Bring New Life to the Brick and Mortar Library

NASA Astrophysics Data System (ADS)

Bosken, S.

2012-08-01

If all the library books and journals can be viewed on your desk top, why come to the physical library? The USNO Library tried to bring the patrons inside the library. One method was to rotate rare book displays each month. As the library holds a fabulous collection of ancient astronomy books, including Copernicus, Kepler, Galileo, and Newton, we have abundant resources. The presentation will highlight the varied displays and offer a Rare Books 101 explanation of paper, printing, binding and a behind-the-scenes look at how old books are maintained and preserved.

Amino acid signature enables proteins to recognize modified tRNA.

PubMed

Spears, Jessica L; Xiao, Xingqing; Hall, Carol K; Agris, Paul F

2014-02-25

Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

Combination of Multiple Spectral Libraries Improves the Current Search Methods Used to Identify Missing Proteins in the Chromosome-Centric Human Proteome Project.

PubMed

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Kim, Kwang-Youl; Kwon, Kyung-Hoon; Yoo, Jong Shin; Omenn, Gilbert S; Baker, Mark S; Hancock, William S; Paik, Young-Ki

2015-12-04

Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of <1% at the peptide and protein levels. The quality-controlled results were then cross-checked with the neXtProt DB (2014-09-19 release). The two spectral libraries, iRefSPL and simSPL, were designed to ensure no overlap of the proteome coverage. They were shown to be complementary to spectral library searching and significantly increased the number of matches. From this trial, 12 new missing proteins were identified that passed the following criterion: at least 2 peptides of 7 or more amino acids in length or one of 9 or more amino acids in length with one or more unique sequences. Thus, the iRefSPL and simSPL combination can be used to help identify peptides that have not been detected by conventional sequence database searches with improved sensitivity and a low error rate.

Peptide Inhibitors of the Amyloidogenesis of IAPP: Verification of the Hairpin Binding Geometry Hypothesis

PubMed Central

Sivanesam, Kalkena; Shu, Irene; Huggins, Kelly N. L.; Tatarek-Nossol, Marianna; Kapurniotu, Aphrodite; Andersen, Niels H.

2016-01-01

Versions of a previously discovered β-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at sub-stoichiometric concentrations relative to this amyloidogenic peptide. The hairpin binding hypothesis stands confirmed. PMID:27317951

Optimization by infusion of multiple reaction monitoring transitions for sensitive quantification of peptides by liquid chromatography/mass spectrometry.

PubMed

Alghanem, Bandar; Nikitin, Frédéric; Stricker, Thomas; Duchoslav, Eva; Luban, Jeremy; Strambio-De-Castillia, Caterina; Muller, Markus; Lisacek, Frédérique; Varesio, Emmanuel; Hopfgartner, Gérard

2017-05-15

In peptide quantification by liquid chromatography/mass spectrometry (LC/MS), the optimization of multiple reaction monitoring (MRM) parameters is essential for sensitive detection. We have compared different approaches to build MRM assays, based either on flow injection analysis (FIA) of isotopically labelled peptides, or on the knowledge and the prediction of the best settings for MRM transitions and collision energies (CE). In this context, we introduce MRMOptimizer, an open-source software tool that processes spectra and assists the user in selecting transitions in the FIA workflow. MS/MS spectral libraries with CE voltages from 10 to 70 V are automatically acquired in FIA mode for isotopically labelled peptides. Then MRMOptimizer determines the optimal MRM settings for each peptide. To assess the quantitative performance of our approach, 155 peptides, representing 84 proteins, were analysed by LC/MRM-MS and the peak areas were compared between: (A) the MRMOptimizer-based workflow, (B1) the SRMAtlas transitions set used 'as-is'; (B2) the same SRMAtlas set with CE parameters optimized by Skyline. 51% of the three most intense transitions per peptide were shown to be common to both A and B1/B2 methods, and displayed similar sensitivity and peak area distributions. The peak areas obtained with MRMOptimizer for transitions sharing either the precursor ion charge state or the fragment ions with the SRMAtlas set at unique transitions were increased 1.8- to 2.3-fold. The gain in sensitivity using MRMOptimizer for transitions with different precursor ion charge state and fragment ions (8% of the total), reaches a ~ 11-fold increase. Isotopically labelled peptides can be used to optimize MRM transitions more efficiently in FIA than by searching databases. The MRMOptimizer software is MS independent and enables the post-acquisition selection of MRM parameters. Coefficients of variation for optimal CE values are lower than those obtained with the SRMAtlas approach (B2) and one additional peptide was detected. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

A novel Chk1-binding peptide that enhances genotoxic sensitivity through the cellular redistribution of nuclear Chk1

PubMed Central

Kim, Kwang Seok; Choi, Kyu Jin; Bae, Sangwoo

2016-01-01

Since checkpoint kinase 1 (Chk1) is an essential factor for cell viability following DNA damage, the inhibition of Chk1 has been a major focus of pharmaceutical development to enhance the sensitivity of tumor cells to chemo- and radiotherapy that damage DNA. However, due to the off-target effects of conventional Chk1-targeting strategies and the toxicity of Chk1 inhibitors, alternative strategies are required to target Chk1. To facilitate such efforts, in this study, we identified a specific Chk1-binding 12-mer peptide from the screening of a phage display library and characterized the peptide in terms of cellular cytotoxicity, and in terms of its effect on Chk1 activity and sensitivity to genotoxic agents. This peptide, named N-terminal Chk1-binding peptide (Chk1-NP), bound the kinase domain of Chk1. Simulation of the binding revealed that the very N-terminus of the Chk1 kinase domain is the potential peptide binding site. Of note, the polyarginine-mediated internalization of Chk1-NP redistributed nuclear Chk1 with a prominent decrease in the nucleus in the absence of DNA damage. Treatment with Chk1-NP peptide alone decreased the viability of p53-defective HeLa cells, but not that of p53-functional NCI-H460 cells under normal conditions. The treatment of HeLa or NCI-H460 cells with the peptide significantly enhanced radiation sensitivity following ionizing radiation (IR) with a greater enhancement observed in HeLa cells. Moreover, the IR-induced destabilization of Chk1 was aggravated by treatment with Chk1-NP. Therefore, the decreased nuclear localization and protein levels of Chk1 seem to be responsible for the enhanced cancer cell killing following combined treatment with IR and Chk1-NP. The approach using the specific Chk1-binding peptide may facilitate the mechanistic understanding and potential modulation of Chk1 activities and may provide a novel rationale for the development of specific Chk1-targeting agents. PMID:28025997

A Liquid Array Platform For the Multiplexed Analysis of Synthetic Molecule-Protein Interactions

PubMed Central

Doran, Todd M.; Kodadek, Thomas

2014-01-01

Synthetic molecule microarrays, consisting of many different compounds spotted onto a planar surface such as modified glass or cellulose, have proven to be useful tools for the multiplexed analysis of small molecule- and peptide-protein interactions. However, these arrays are technically difficult to manufacture and use with high reproducibility and require specialized equipment. Here we report a more convenient alternative comprised of color-encoded beads that display a small molecule protein ligand on the surface. Quantitative, multiplexed assay of protein binding to up to 24 different ligands can be achieved using a common flow cytometer for the readout. This technology should be useful for evaluating hits from library screening efforts, the determination of structure activity relationships and for certain types of serological analyses. PMID:24245981

CD68 acts as a major gateway for malaria sporozoite liver infection

PubMed Central

Cha, Sung-Jae; Park, Kiwon; Srinivasan, Prakash; Schindler, Christian W.; van Rooijen, Nico; Stins, Monique

2015-01-01

After being delivered by the bite from an infected mosquito, Plasmodium sporozoites enter the blood circulation and infect the liver. Previous evidence suggests that Kupffer cells, a macrophage-like component of the liver blood vessel lining, are traversed by sporozoites to initiate liver invasion. However, the molecular determinants of sporozoite–Kupffer cell interactions are unknown. Understanding the molecular basis for this specific recognition may lead to novel therapeutic strategies to control malaria. Using a phage display library screen, we identified a peptide, P39, that strongly binds to the Kupffer cell surface and, importantly, inhibits sporozoite Kupffer cell entry. Furthermore, we determined that P39 binds to CD68, a putative receptor for sporozoite invasion of Kupffer cells that acts as a gateway for malaria infection of the liver. PMID:26216124

ORFeome Phage Display.

PubMed

Zantow, Jonas; Moreira, Gustavo Marçal Schmidt Garcia; Dübel, Stefan; Hust, Michael

2018-01-01

ORFeome phage display allows the efficient functional screening of entire proteomes or even metaproteomes to identify immunogenic proteins. For this purpose, randomly fragmented, whole genomes or metagenomes are cloned into a phage-display vector allowing positive selection for open reading frames (ORF) to improve the library quality. These libraries display all possible proteins encoded by a pathogen or a microbiome on the phage surface. Consequently, immunogenic proteins can be selected from these libraries using disease-related immunoglobulins from patient serum. ORFeome phage display in particular allows the identification of immunogenic proteins that are only expressed in the host-pathogen interaction but not in cultivation, as well as the detection of very low expressed and very small immunogens and immunogenic proteins of non-cultivable organisms. The identified immunogenic proteins are potential biomarkers for the development of diagnostic assays or vaccines. These articles will give an introduction to ORFeome phage-display technology and give detailed protocols to identify immunogenic proteins by phage display.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

PubMed

Xu, Hai; Bao, Xi; Wang, Yiwei; Xu, Yue; Deng, Bihua; Lu, Yu; Hou, Jibo

2018-03-20

DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Exploring the active site binding specificity of kallikrein-related peptidase 5 (KLK5) guides the design of new peptide substrates and inhibitors.

PubMed

de Veer, Simon J; Swedberg, Joakim E; Brattsand, Maria; Clements, Judith A; Harris, Jonathan M

2016-12-01

Kallikrein-related peptidase 5 (KLK5) is a promising therapeutic target in several skin diseases, including Netherton syndrome, and is emerging as a potential target in various cancers. In this study, we used a sparse matrix library of 125 individually synthesized peptide substrates to characterize the binding specificity of KLK5. The sequences most favored by KLK5 were GRSR, YRSR and GRNR, and we identified sequence-specific interactions involving the peptide N-terminus by analyzing kinetic constants (kcat and KM) and performing molecular dynamics simulations. KLK5 inhibitors were subsequently engineered by substituting substrate sequences into the binding loop (P1, P2 and P4 residues) of sunflower trypsin inhibitor-1 (SFTI-1). These inhibitors were effective against KLK5 but showed limited selectivity, and performing a further substitution at P2' led to the design of a new variant that displayed improved activity against KLK5 (Ki=4.2±0.2 nm), weak activity against KLK7 and 12-fold selectivity over KLK14. Collectively, these findings provide new insight into the design of highly favored binding sequences for KLK5 and reveal several opportunities for modulating inhibitor selectivity over closely related proteases that will be useful for future studies aiming to develop therapeutic molecules targeting KLK5.

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.

1987-06-01

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from lambdagt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. Inmore » RNA blots of poly(A)/sup +/ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.« less

Peptide Inhibitors of the amyloidogenesis of IAPP: verification of the hairpin-binding geometry hypothesis.

PubMed

Sivanesam, Kalkena; Shu, Irene; Huggins, Kelly N L; Tatarek-Nossol, Marianna; Kapurniotu, Aphrodite; Andersen, Niels H

2016-08-01

Versions of a previously discovered β-hairpin peptide inhibitor of IAPP aggregation that are stabilized in that conformation, or even forced to remain in the hairpin conformation by a backbone cyclization constraint, display superior activity as inhibitors. The cyclized hairpin, cyclo-WW2, displays inhibitory activity at substoichiometric concentrations relative to this amyloidogenic peptide. The hairpin-binding hypothesis stands confirmed. © 2016 Federation of European Biochemical Societies.

Identification of a Novel Lysosomal Trafficking Peptide using Phage Display Biopanning Coupled with Endocytic Selection Pressure

PubMed Central

2015-01-01

Methods to select ligands that accumulate specifically in cancer cells and traffic through a defined endocytic pathway may facilitate rapid pairing of ligands with linkers suitable for drug conjugate therapies. We performed phage display biopanning on cancer cells that are treated with selective inhibitors of a given mechanism of endocytosis. Using chlorpromazine to inhibit clathrin-mediated endocytosis in H1299 nonsmall cell lung cancer cells, we identified two clones, ATEPRKQYATPRVFWTDAPG (15.1) and a novel peptide LQWRRDDNVHNFGVWARYRL (H1299.3). The peptides segregate by mechanism of endocytosis and subsequent location of subcellular accumulation. The H1299.3 peptide primarily utilizes clathrin-mediated endocytosis and colocalizes with Lamp1, a lysosomal marker. Conversely, the 15.1 peptide is clathrin-independent and localizes to a perinuclear region. Thus, this novel phage display scheme allows for selection of peptides that selectively internalize into cells via a known mechanism of endocytosis. These types of selections may allow for better matching of linker with targeting ligand by selecting ligands that internalize and traffic to known subcellular locations. PMID:25188559

Peptidomics combined with cDNA library unravel the diversity of centipede venom.

PubMed

Rong, Mingqiang; Yang, Shilong; Wen, Bo; Mo, Guoxiang; Kang, Di; Liu, Jie; Lin, Zhilong; Jiang, Wenbin; Li, Bowen; Du, Chaoqin; Yang, Shuanjuan; Jiang, Hui; Feng, Qiang; Xu, Xun; Wang, Jun; Lai, Ren

2015-01-30

Centipedes are one of the oldest venomous arthropods using toxin as their weapon to capture prey. But little attention was focused on them and only few centipede toxins were demonstrated with activity on ion channels. Therefore, more deep works are needed to understand the diversity of centipede venom. In the present study, we use peptidomics combined with cDNA library to uncover the diversity of centipede Scolopendra subspinipes mutilans L. Koch. 192 peptides were identified by LC-MS/MS and 79 precursors were deduced by cDNA library. Surprisingly, the signal peptides of centipede toxins were more complicated than any other animal toxins and even exhibited large differences in homologues. Meanwhile, a large number of variants generated by alternative cleavage sites were detected by mass spectra. Odd number of cystein (3, 5, 7) found in the mature peptides were seldom seen in peptide toxins. In additional, two novel cysteine frameworks (C-C-C-CCC, C-C-C-C-CC-CC) were identified from 16 different cysteine frameworks from centipede peptides. Only 29 precursors have clear targets, while others may provide a potential diversity function for centipede. These findings highlight the extensive diversity of centipede toxins and provide powerful tools to understand the capture and defense weapon of centipede. Peptide toxins from venomous animal have attracted increasing attentions due to their extraordinary chemical and pharmacological diversity. Centipedes are one of the most used Chinese traditional medicines, but little was known about the active components. The venom of Scolopendra subspinipes mutilans L. Koch is first deeply analyzed in this work and most of peptides were never discovered before. Interestingly, the number and arrangement of cysteine showed a larger different to known peptide toxins such spider or scorpion toxins. Moreover, only 29 peptides from this centipede venom were identified with known function. It suggested that our work not only important to understand the composition of centipede venom, but also provide many valuable peptides for potential biological functions. Copyright © 2014 Elsevier B.V. All rights reserved.

Arming Technology in Yeast-Novel Strategy for Whole-cell Biocatalyst and Protein Engineering.

PubMed

Kuroda, Kouichi; Ueda, Mitsuyoshi

2013-09-09

Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called "arming technology", can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach.

Identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a QCM device.

PubMed

Nishiyama, Kazusa; Takakusagi, Yoichi; Kusayanagi, Tomoe; Matsumoto, Yuki; Habu, Shiori; Kuramochi, Kouji; Sugawara, Fumio; Sakaguchi, Kengo; Takahashi, Hideyo; Natsugari, Hideaki; Kobayashi, Susumu

2009-01-01

Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo.

Application Reuse Library for Software, Requirements, and Guidelines

NASA Technical Reports Server (NTRS)

Malin, Jane T.; Thronesbery, Carroll

1994-01-01

Better designs are needed for expert systems and other operations automation software, for more reliable, usable and effective human support. A prototype computer-aided Application Reuse Library shows feasibility of supporting concurrent development and improvement of advanced software by users, analysts, software developers, and human-computer interaction experts. Such a library expedites development of quality software, by providing working, documented examples, which support understanding, modification and reuse of requirements as well as code. It explicitly documents and implicitly embodies design guidelines, standards and conventions. The Application Reuse Library provides application modules with Demo-and-Tester elements. Developers and users can evaluate applicability of a library module and test modifications, by running it interactively. Sub-modules provide application code and displays and controls. The library supports software modification and reuse, by providing alternative versions of application and display functionality. Information about human support and display requirements is provided, so that modifications will conform to guidelines. The library supports entry of new application modules from developers throughout an organization. Example library modules include a timer, some buttons and special fonts, and a real-time data interface program. The library prototype is implemented in the object-oriented G2 environment for developing real-time expert systems.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses

PubMed Central

Rucevic, Marijana; Kourjian, Georgio; Boucau, Julie; Blatnik, Renata; Garcia Bertran, Wilfredo; Berberich, Matthew J.; Walker, Bruce D.; Riemer, Angelika B.

2016-01-01

ABSTRACT Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity. IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity. PMID:27440904

Rapid, Multiplexed Microfluidic Phage Display

DTIC Science & Technology

2012-01-01

affinity phage- displayed peptides for multiple targets in just a single round and without the need for bacterial infection. The chip is shown to be able...by bacterial titer and amplification, and at least two additional rounds of selection. After the final round of biopan- ning, eluted phage are grown on...agar plates, and individual plaques are selected for DNA characterization to determine the amino acid sequence of the phage-displayed peptides. While

Using the ribosome to synthesize peptidomimetics

PubMed Central

2009-01-01

Peptidomimetic research is an approach to identify peptide-based drugs designed to mimic structural, conformational, and biological properties of peptides while overcoming their limitations, such as protease instability and poor cell penetration. With recent advances in ribosomal synthesis of peptides containing unnatural amino acids, this technology appears suitable for preparing large structurally diverse libraries of peptidomimetics for drug discovery screening. PMID:20948631

Advances in Synthetic Peptides Reagent Discovery

DTIC Science & Technology

2013-07-01

to promote specific and high affinity binding. Longer incubations may result in nonspecific attachment, such as early biofilm formation. Because...peptide libraries yields ligand arrays that classify breast tumor subtypes,” Molecular Cancer Therapeutics, 8(5), 1312-1318 (2009). [26] J. M. Kogot

De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs.

PubMed

Menegatti, Stefano; Zakrewsky, Michael; Kumar, Sunny; De Oliveira, Joshua Sanchez; Muraski, John A; Mitragotri, Samir

2016-03-09

Skin-penetrating peptides (SPPs) are attracting increasing attention as a non-invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effective enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide-cyclic peptides against keratin and cyclosporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA permeation on par with previously identified SPP SPACE (TM) . An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also validated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin-penetrating peptides for the delivery of desired therapeutic or cosmetic agents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tailoring gas sensor arrays via the design of short peptides sequences as binding elements.

PubMed

Mascini, Marcello; Pizzoni, Daniel; Perez, German; Chiarappa, Emilio; Di Natale, Corrado; Pittia, Paola; Compagnone, Dario

2017-07-15

A semi-combinatorial virtual approach was used to prepare peptide-based gas sensors with binding properties towards five different chemical classes (alcohols, aldehydes, esters, hydrocarbons and ketones). Molecular docking simulations were conducted for a complete tripeptide library (8000 elements) versus 58 volatile compounds belonging to those five chemical classes. By maximizing the differences between chemical classes, a subset of 120 tripeptides was extracted and used as scaffolds for generating a combinatorial library of 7912 tetrapeptides. This library was processed in an analogous way to the former. Five tetrapeptides (IHRI, KSDS, LGFD, TGKF and WHVS) were chosen depending on their virtual affinity and cross-reactivity for the experimental step. The five peptides were covalently bound to gold nanoparticles by adding a terminal cysteine to each tetrapeptide and deposited onto 20MHz quartz crystal microbalances to construct the gas sensors. The behavior of peptides after this chemical modification was simulated at the pH range used in the immobilization step. ΔF signals analyzed by principal component analysis matched the virtually screened data. The array was able to clearly discriminate the 13 volatile compounds tested based on their hydrophobicity and hydrophilicity molecules as well as the molecular weight. Copyright © 2016 Elsevier B.V. All rights reserved.

The New Visual Displays That Are "Floating" Your Way. Building Digital Libraries

ERIC Educational Resources Information Center

Huwe, Terence K.

2005-01-01

In this column, the author describes three very experimental visual display technologies that will affect library collections and services in the near future. While each of these new display strategies is unique in its technological approach, there is a common denominator to all three: better freedom of mobility that will allow people to interact…

Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

PubMed Central

Martínez-Arteaga, Rocio; Ruano-Gallego, David; Fraile, Sofía; Margolles, Yago; Teira, Xema; Gutierrez, Carlos; Bodelón, Gustavo; Fernández, Luis Ángel

2013-01-01

Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions. PMID:24086454

Rapid isolation of novel FK506 binding proteins from multiple organisms using gDNA and cDNA T7 phage display.

PubMed

Piggott, Andrew M; Kriegel, Alison M; Willows, Robert D; Karuso, Peter

2009-10-01

Reverse chemical proteomics using T7 phage display is a powerful technique for identifying cellular receptors of biologically active small molecules. However, to date this method has generally been limited to cDNA libraries constructed from mRNA isolated from eukaryotes. In this paper, we describe the construction of the first prokaryotic T7 phage display libraries from randomly digested Pseudomonas stutzeri and Vibrio fischeri gDNA, as well as a plant cDNA library from Arabidopsis thaliana. We also describe the use of T7 phage display to identify novel proteins from environmental DNA samples using biotinylated FK506 as a model affinity probe.

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

PubMed

Houimel, Mehdi; Mazzucchelli, Luca

2013-01-01

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

DOE PAGES

Sun, Yue; Ban, Bhupal; Bradbury, Andrew; ...

2016-08-29

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

Combining yeast display and competitive FACS to select rare hapten-specific clones from recombinant antibody libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Yue; Ban, Bhupal; Bradbury, Andrew

The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10 6 ) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used firstmore » to enrich the library between 20- and 100- fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. Furthermore, this selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.« less

An interleukin 13 receptor α 2–specific peptide homes to human Glioblastoma multiforme xenografts

PubMed Central

Pandya, Hetal; Gibo, Denise M.; Garg, Shivank; Kridel, Steven; Debinski, Waldemar

2012-01-01

Interleukin 13 receptor α 2 (IL-13Rα2) is a glioblastoma multiforme (GBM)–associated plasma membrane receptor, a brain tumor of dismal prognosis. Here, we isolated peptide ligands for IL-13Rα2 with use of a cyclic disulphide-constrained heptapeptide phages display library and 2 in vitro biopanning schemes with GBM cells that do (G26-H2 and SnB19-pcDNA cells) or do not (G26-V2 and SnB19-asIL-13Rα2 cells) over-express IL-13Rα2. We identified 3 peptide phages that bind to IL-13Rα2 in cellular and protein assays. One of the 3 peptide phages, termed Pep-1, bound to IL-13Rα2 with the highest specificity, surprisingly, also in a reducing environment. Pep-1 was thus synthesized and further analyzed in both linear and disulphide-constrained forms. The linear peptide bound to IL-13Rα2 more avidly than did the disulphide-constrained form and was efficiently internalized by IL-13Rα2–expressing GBM cells. The native ligand, IL-13, did not compete for the Pep-1 binding to the receptor and vice versa in any of the assays, indicating that the peptide might be binding to a site on the receptor different from the native ligand. Furthermore, we demonstrated by noninvasive near infrared fluorescence imaging in nude mice that Pep-1 binds and homes to both subcutaneous and orthotopic human GBM xenografts expressing IL-13Rα2 when injected by an intravenous route. Thus, we identified a linear heptapeptide specific for the IL-13Rα2 that is capable of crossing the blood-brain tumor barrier and homing to tumors. Pep-1 can be further developed for various applications in cancer and/or inflammatory diseases. PMID:21946118

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078