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.

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.

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.

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.

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.

[Preparation and activity validation of PP7 bacteriophage-like particles displaying PAP114-128 peptide].

PubMed

Sun, Yanli; Sun, Yanhua

2016-10-01

Objective To obtain the PP7 bacteriophage-like particles carrying the peptide of prostatic acid phosphatase PAP 114-128 , and prove that they retain the original biological activity. Methods First, the plasmid pETDuet-2PP7 was constructed as follows: the gene of PP7 coat protein dimer was amplified by gene mutation combined with overlapping PCR technology, and inserted into the vector pETDuet-1. Following that, the plasmid pETDuet-2PP7-PAP 114-128 was constructed as follows: the PP7 coat protein gene carrying the coding gene of PAP 114-128 peptide was amplified using PCR, and then inserted into the vector pETDuet-2PP7. Both pETDuet-2PP7 and pETDuet-2PP7-PAP 114-128 were transformed into E.coli and expressed. The expression product was verified by SDS-PAGE, double immunodiffusion assay and ELISA. Results The gene fragment of PP7 coat protein dimer was obtained by overlapping PCR using Ex Taq DNA polymerase, and the antigenicity of its expression product was the same as that of the coat protein of wild-type PP7 bacteriophage. Moreover, the PAP 114-128 peptide epitope that was displayed on the surface of PP7 bacteriophage was identical with the corresponding epitope of natural human PAP, and it was able to induce high levels of antibodies. Conclusion The gene of PP7 coat protein dimer with repeated sequences can be prepared by gene mutation combined with overlapping PCR. Based on this, PP7 bacteriophage-like particles carrying PAP peptide can be prepared, which not only solves the problem of the instability of the peptides, but also lays a foundation for the study on their delivery and function.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Structural analysis and insertion study reveal the ideal sites for surface displaying foreign peptides on a betanodavirus-like particle.

PubMed

Xie, Junfeng; Li, Kunpeng; Gao, Yuanzhu; Huang, Runqing; Lai, Yuxiong; Shi, Yan; Yang, Shaowei; Zhu, Guohua; Zhang, Qinfen; He, Jianguo

2016-01-11

Betanodavirus infection causes fatal disease of viral nervous necrosis in many cultured marine and freshwater fish worldwide and the virus-like particles (VLP) are effective vaccines against betanodavirus. But vaccine and viral vector designs of betanodavirus VLP based on their structures remain lacking. Here, the three-dimensional structure of orange-spotted grouper nervous necrosis virus (OGNNV) VLP (RBS) at 3.9 Å reveals the organization of capsid proteins (CP). Based on the structural results, seven putative important sites were selected to genetically insert a 6× histidine (His)-tag for VLP formation screen, resulting in four His-tagged VLP (HV) at positions N-terminus, Ala220, Pro292 and C-terminus. The His-tags of N-terminal HV (NHV) were concealed inside virions while those of 220HV and C-terminal HV (CHV) were displayed at the outer surface. NHV, 220HV and CHV maintained the same cell entry ability as RBS in the Asian sea bass (SB) cell line, indicating that their similar surface structures can be recognized by the cellular entry receptor(s). For application of vaccine design, chromatography-purified CHV could provoke NNV-specific antibody responses as strong as those of RBS in a sea bass immunization assay. Furthermore, in carrying capacity assays, N-terminus and Ala220 can only carry short peptides and C-terminus can even accommodate large protein such as GFP to generate fluorescent VLP (CGV). For application of a viral vector, CGV could be real-time visualized to enter SB cells in invasion study. All the results confirmed that the C-terminus of CP is a suitable site to accommodate foreign peptides for vaccine design and viral vector development.

Trastuzumab-binding peptide display by Tobacco mosaic virus

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

Frolova, Olga Y.; Petrunia, Igor V.; Komarova, Tatiana V.

2010-11-10

Human epidermal growth factor receptor-2 (HER2/neu) is a target for the humanized monoclonal antibody trastuzumab. Recently, trastuzumab-binding peptides (TBP) of HER2/neu that inhibit proliferation of breast cancer cells were identified. We have now studied conditions of efficient assembly in vivo of Tobacco mosaic virus (TMV)-based particles displaying TBP on its surface. The system is based on an Agrobacterium-mediated co-delivery of binary vectors encoding TMV RNA and coat protein (CP) with TBP in its C-terminal extension into plant leaves. We show how the fusion of amino acid substituted TBP (sTBP) to CP via a flexible peptide linker can improve the manufacturabilitymore » of recombinant TMV (rTMV). We also reveal that rTMV particles with exposed sTBP retained trastuzumab-binding capacity but lost an anti-HER2/neu immunogenic scaffold function. Mouse antibodies against rTMV did not recognize HER2/neu on surface of human SK-BR-3 cells.« less

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.

Plectin-1 Targeted AAV Vector for the Molecular Imaging of Pancreatic Cancer

PubMed Central

Konkalmatt, Prasad R.; Deng, Defeng; Thomas, Stephanie; Wu, Michael T.; Logsdon, Craig D.; French, Brent A.; Kelly, Kimberly A.

2013-01-01

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant disease that is the fourth leading cause of cancer-related death in the US. Gene therapy using AAV vectors to selectively deliver genes to PDAC cells is an attractive treatment option for pancreatic cancer. However, most AAV serotypes display a broad spectrum of tissue tropism and none of the existing serotypes specifically target PDAC cells. This study tests the hypothesis that AAV2 can be genetically re-engineered to specifically target PDAC cells by modifying the capsid surface to display a peptide that has previously been shown to bind plectin-1. Toward this end, a Plectin-1 Targeting Peptide (PTP) was inserted into the loop IV region of the AAV2 capsid, and the resulting capsid (AAV-PTP) was used in a series of in vitro and in vivo experiments. In vitro, AAV-PTP was found to target all five human PDAC cell lines tested (PANC-1, MIA PaCa-2, HPAC, MPanc-96, and BxPC-3) preferentially over two non-neoplastic human pancreatic cell lines (human pancreatic ductal epithelial and human pancreatic stellate cells). In vivo, mice bearing subcutaneous tumor xenografts were generated using the PANC-1 cell line. Once tumors reached a size of ∼1–2 mm in diameter, the mice were injected intravenously with luciferase reporter vectors packaged in the either AAV-PTP or wild type AAV2 capsids. Luciferase expression was then monitored by bioluminescence imaging on days 3, 7, and 14 after vector injection. The results indicate that the AAV-PTP capsid displays a 37-fold preference for PANC-1 tumor xenographs over liver and other tissues; whereas the wild type AAV2 capsid displays a complementary preference for liver over tumors and other tissues. Together, these results establish proof-of-principle for the ability of PTP-modified AAV capsids to selectively target gene delivery to PDAC cells in vivo, which opens promising new avenues for the early detection, diagnosis, and treatment of pancreatic cancer. PMID:23616947

A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

PubMed

Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

2015-09-01

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

A receptor-targeted nanocomplex vector system optimized for respiratory gene transfer.

PubMed

Tagalakis, Aristides D; McAnulty, Robin J; Devaney, James; Bottoms, Stephen E; Wong, John B; Elbs, Martin; Writer, Michele J; Hailes, Helen C; Tabor, Alethea B; O'Callaghan, Christopher; Jaffe, Adam; Hart, Stephen L

2008-05-01

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.

Machine learning study for the prediction of transdermal peptide

NASA Astrophysics Data System (ADS)

Jung, Eunkyoung; Choi, Seung-Hoon; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Yun-Jaie; Shin, Jae-Min; Choi, Kihang; Jung, Dong Hyun

2011-04-01

In order to develop a computational method to rapidly evaluate transdermal peptides, we report approaches for predicting the transdermal activity of peptides on the basis of peptide sequence information using Artificial Neural Network (ANN), Partial Least Squares (PLS) and Support Vector Machine (SVM). We identified 269 transdermal peptides by the phage display technique and use them as the positive controls to develop and test machine learning models. Combinations of three descriptors with neural network architectures, the number of latent variables and the kernel functions are tried in training to make appropriate predictions. The capacity of models is evaluated by means of statistical indicators including sensitivity, specificity, and the area under the receiver operating characteristic curve (ROC score). In the ROC score-based comparison, three methods proved capable of providing a reasonable prediction of transdermal peptide. The best result is obtained by SVM model with a radial basis function and VHSE descriptors. The results indicate that it is possible to discriminate between transdermal peptides and random sequences using our models. We anticipate that our models will be applicable to prediction of transdermal peptide for large peptide database for facilitating efficient transdermal drug delivery through intact skin.

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

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.

Physicochemical and immunological characterization of chitosan-coated bacteriophage nanoparticles for in vivo mycotoxin modeling.

PubMed

de Andrade, Carla Yoko Tanikawa; Yamanaka, Isabel; Schlichta, Laís S; Silva, Sabrina Karim; Picheth, Guilherme F; Caron, Luiz Felipe; de Moura, Juliana; de Freitas, Rilton Alves; Alvarenga, Larissa Magalhães

2018-04-01

To propose a novel modeling of aflatoxin immunization and surrogate toxin conjugate from AFB1 vaccines, an immunogen based on the mimotope, (i.e. a peptide-displayed phage that mimics aflatoxins epitope without toxin hazards) was designed. The recombinant phage 3P30 was identified by phage display technology and exhibited the ability to bind, dose dependent, specifically to its cognate target - anti-AFB1 antibody. In immunization assay, the phage-displayed mimotope and its peptide chemically synthesized were able to induce specific anti-AFB1 antibodies, indicating the proof of concept for aflatoxin mimicry. Furthermore, the phage 3P30 was homogeneously coated with chitosan, which also provided a tridimensional matrix network for mucosal delivery. After intranasal immunization, chitosan coated phages improved specific immunogenicity compared to the free antigen. It can be concluded that affinity-selected phage may contribute to the rational design of epitope-based vaccines in a prospectus for the control of aflatoxins and possibly other mycotoxins, and that chitosan coating improved the vectorization of the vaccine by the mucosal route. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chloroplast-expressed MSI-99 in tobacco improves disease resistance and displays inhibitory effect against rice blast fungus.

PubMed

Wang, Yun-Peng; Wei, Zheng-Yi; Zhang, Yu-Ying; Lin, Chun-Jing; Zhong, Xiao-Fang; Wang, Yue-Lin; Ma, Jing-Yong; Ma, Jian; Xing, Shao-Chen

2015-03-02

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.

Design and construction of 2A peptide-linked multicistronic vectors.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Method for predicting peptide detection in mass spectrometry

DOEpatents

Kangas, Lars [West Richland, WA; Smith, Richard D [Richland, WA; Petritis, Konstantinos [Richland, WA

2010-07-13

A method of predicting whether a peptide present in a biological sample will be detected by analysis with a mass spectrometer. The method uses at least one mass spectrometer to perform repeated analysis of a sample containing peptides from proteins with known amino acids. The method then generates a data set of peptides identified as contained within the sample by the repeated analysis. The method then calculates the probability that a specific peptide in the data set was detected in the repeated analysis. The method then creates a plurality of vectors, where each vector has a plurality of dimensions, and each dimension represents a property of one or more of the amino acids present in each peptide and adjacent peptides in the data set. Using these vectors, the method then generates an algorithm from the plurality of vectors and the calculated probabilities that specific peptides in the data set were detected in the repeated analysis. The algorithm is thus capable of calculating the probability that a hypothetical peptide represented as a vector will be detected by a mass spectrometry based proteomic platform, given that the peptide is present in a sample introduced into a mass spectrometer.

Design and construction of targeted AAVP vectors for mammalian cell transduction.

PubMed

Hajitou, Amin; Rangel, Roberto; Trepel, Martin; Soghomonyan, Suren; Gelovani, Juri G; Alauddin, Mian M; Pasqualini, Renata; Arap, Wadih

2007-01-01

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

Expression of a foot-and-mouth disease virus immunodominant epitope by a filamentous bacteriophage vector.

PubMed

Kim, Y J; Lebreton, F; Kaiser, C; Crucière, C; Rémond, M

2004-02-01

We described the construction of a recombinant filamentous phage displaying on its surface the immunodominant site of VP1 protein of foot-and-mouth disease virus (FMDV). The coding sequence was inserted at the amino-terminus of the major coat protein pVIII via a spacer. The hybrid phage proved to be antigenic as it was recognized by polyclonal and monoclonal anti FMDV sera. In two experiments involving immunisation of guinea-pigs with the recombinant phage, a low antibody response was generated. This suggests a possible role for phage displayed peptides in inducing anti FMDV immunity and the possibility of further development is discussed.

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

PubMed Central

EL Andaloussi, Samir; Lehto, Taavi; Mäger, Imre; Rosenthal-Aizman, Katri; Oprea, Iulian I.; Simonson, Oscar E.; Sork, Helena; Ezzat, Kariem; Copolovici, Dana M.; Kurrikoff, Kaido; Viola, Joana R.; Zaghloul, Eman M.; Sillard, Rannar; Johansson, Henrik J.; Said Hassane, Fatouma; Guterstam, Peter; Suhorutšenko, Julia; Moreno, Pedro M. D.; Oskolkov, Nikita; Hälldin, Jonas; Tedebark, Ulf; Metspalu, Andres; Lebleu, Bernard; Lehtiö, Janne; Smith, C. I. Edvard; Langel, Ülo

2011-01-01

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential. PMID:21245043

On The Influence Of Vector Design On Antibody Phage Display

PubMed Central

Soltes, Glenn; Hust, Michael; Ng, Kitty K.Y.; Bansal, Aasthaa; Field, Johnathan; Stewart, Donald I.H.; Dübel, Stefan; Cha, Sanghoon; Wiersma, Erik J

2007-01-01

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection. PMID:16996161

On the influence of vector design on antibody phage display.

PubMed

Soltes, Glenn; Hust, Michael; Ng, Kitty K Y; Bansal, Aasthaa; Field, Johnathan; Stewart, Donald I H; Dübel, Stefan; Cha, Sanghoon; Wiersma, Erik J

2007-01-20

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.

[New strategy for RNA vectorization in mammalian cells. Use of a peptide vector].

PubMed

Vidal, P; Morris, M C; Chaloin, L; Heitz, F; Divita, G

1997-04-01

A major barrier for gene delivery is the low permeability of nucleic acids to cellular membranes. The development of antisenses and gene therapy has focused mainly on improving methods of oligonucleotide or gene delivery to the cell. In this report we described a new strategy for RNA cell delivery, based on a short single peptide. This peptide vector is derived from both the fusion domain of the gp41 protein of HIV and the nuclear localization sequence of the SV40 large T antigen. This peptide vector localizes rapidly to the cytoplasm then to the nucleus of human fibroblasts (HS-68) within a few minutes and exhibits a high affinity for a single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase (in a 100 nM range). The peptide/RNA complex formation involves mainly electrostatic interactions between the basic residues of the peptide and the charges on the phosphate group of the RNA. In the presence of the peptide-vector fluorescently-labelled mRNA is delivered into the cytoplasm of mammalian cells (HS68 human fibroblasts) in less than 1 h with a relatively high efficiency (80%). This new concept based on a peptide-derived vector offers several advantages compared to other compounds commonly used in gene delivery. This vector is highly soluble and exhibits no cytotoxicity at the concentrations used for optimal gene delivery. This result clearly supports the fact that this peptide vector is a powerful tool and that it can be used widely, as much for laboratory research as for new applications and development in gene and/or antisense therapy.

Advancement and applications of peptide phage display technology in biomedical science.

PubMed

Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

2016-01-19

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Method for enhanced accuracy in predicting peptides using liquid separations or chromatography

DOEpatents

Kangas, Lars J.; Auberry, Kenneth J.; Anderson, Gordon A.; Smith, Richard D.

2006-11-14

A method for predicting the elution time of a peptide in chromatographic and electrophoretic separations by first providing a data set of known elution times of known peptides, then creating a plurality of vectors, each vector having a plurality of dimensions, and each dimension representing the elution time of amino acids present in each of these known peptides from the data set. The elution time of any protein is then be predicted by first creating a vector by assigning dimensional values for the elution time of amino acids of at least one hypothetical peptide and then calculating a predicted elution time for the vector by performing a multivariate regression of the dimensional values of the hypothetical peptide using the dimensional values of the known peptides. Preferably, the multivariate regression is accomplished by the use of an artificial neural network and the elution times are first normalized using a transfer function.

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.

Surface coating of siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers: enhanced gene silencing and reduced adverse effects in vitro

NASA Astrophysics Data System (ADS)

Zeng, Xianghui; de Groot, Anne Marit; Sijts, Alice J. A. M.; Broere, Femke; Oude Blenke, Erik; Colombo, Stefano; van Eden, Willem; Franzyk, Henrik; Nielsen, Hanne Mørck; Foged, Camilla

2015-11-01

Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/β-peptoid peptidomimetics with a systematically varied number of repeating lysine and homoarginine residues was shown to self-assemble with small interfering RNA (siRNA). The resulting well-defined nanocomplexes were coated with anionic lipids giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA-peptidomimetic nanocomplex core of the liposomes, which facilitated siRNA release. Additionally, the optimal anionic liposomes showed efficient intracellular uptake and endosomal escape. Therefore, these findings suggest that a more efficacious and safe formulation can be achieved by surface coating of the siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers.Cationic vectors have demonstrated the potential to facilitate intracellular delivery of therapeutic oligonucleotides. However, enhanced transfection efficiency is usually associated with adverse effects, which also proves to be a challenge for vectors based on cationic peptides. In this study a series of proteolytically stable palmitoylated α-peptide/β-peptoid peptidomimetics with a systematically varied number of repeating lysine and homoarginine residues was shown to self-assemble with small interfering RNA (siRNA). The resulting well-defined nanocomplexes were coated with anionic lipids giving rise to net anionic liposomes. These complexes and the corresponding liposomes were optimized towards efficient gene silencing and low adverse effects. The optimal anionic liposomes mediated a high silencing effect, which was comparable to that of the control (cationic Lipofectamine 2000), and did not display any noticeable cytotoxicity and immunogenicity in vitro. In contrast, the corresponding nanocomplexes mediated a reduced silencing effect with a more narrow safety window. The surface coating with anionic lipid bilayers led to partial decomplexation of the siRNA-peptidomimetic nanocomplex core of the liposomes, which facilitated siRNA release. Additionally, the optimal anionic liposomes showed efficient intracellular uptake and endosomal escape. Therefore, these findings suggest that a more efficacious and safe formulation can be achieved by surface coating of the siRNA-peptidomimetic nano-self-assemblies with anionic lipid bilayers. Electronic supplementary information (ESI) available: Non-fusogenic liposomes; cytotoxicity of naked siRNA and the empty vector; immunogenicity; low-magnification images; DOPE/DPPC liposomes. See DOI: 10.1039/c5nr04807a

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

Bacteriophages as scaffolds for bipartite display: designing swiss army knives on a nanoscale.

PubMed

Molek, Peter; Bratkovič, Tomaž

2015-03-18

Bacteriophages have been exploited as cloning vectors and display vehicles for decades owing to their genetic and structural simplicity. In bipartite display setting, phage takes on the role of a handle to which two modules are attached, each endowing it with specific functionality, much like the Swiss army knife. This concept offers unprecedented potential for phage applications in nanobiotechnology. Here, we compare common phage display platforms and discuss approaches to simultaneously append two or more different (poly)peptides or synthetic compounds to phage coat using genetic fusions, chemical or enzymatic conjugations, and in vitro noncovalent decoration techniques. We also review current reports on design of phage frameworks to link multiple effectors, and their use in diverse scientific disciplines. Bipartite phage display had left its mark in development of biosensors, vaccines, and targeted delivery vehicles. Furthermore, multifunctionalized phages have been utilized to template assembly of inorganic materials and protein complexes, showing promise as scaffolds in material sciences and structural biology, respectively.

Intra-domain phage display (ID-PhD) of peptides and protein mini-domains censored from canonical pIII phage display.

PubMed

Tjhung, Katrina F; Deiss, Frédérique; Tran, Jessica; Chou, Ying; Derda, Ratmir

2015-01-01

In this paper, we describe multivalent display of peptide and protein sequences typically censored from traditional N-terminal display on protein pIII of filamentous bacteriophage M13. Using site-directed mutagenesis of commercially available M13KE phage cloning vector, we introduced sites that permit efficient cloning using restriction enzymes between domains N1 and N2 of the pIII protein. As infectivity of phage is directly linked to the integrity of the connection between N1 and N2 domains, intra-domain phage display (ID-PhD) allows for simple quality control of the display and the natural variations in the displayed sequences. Additionally, direct linkage to phage propagation allows efficient monitoring of sequence cleavage, providing a convenient system for selection and evolution of protease-susceptible or protease-resistant sequences. As an example of the benefits of such an ID-PhD system, we displayed a negatively charged FLAG sequence, which is known to be post-translationally excised from pIII when displayed on the N-terminus, as well as positively charged sequences which suppress production of phage when displayed on the N-terminus. ID-PhD of FLAG exhibited sub-nanomolar apparent Kd suggesting multivalent nature of the display. A TEV-protease recognition sequence (TEVrs) co-expressed in tandem with FLAG, allowed us to demonstrate that 99.9997% of the phage displayed the FLAG-TEVrs tandem and can be recognized and cleaved by TEV-protease. The residual 0.0003% consisted of phage clones that have excised the insert from their genome. ID-PhD is also amenable to display of protein mini-domains, such as the 33-residue minimized Z-domain of protein A. We show that it is thus possible to use ID-PhD for multivalent display and selection of mini-domain proteins (Affibodies, scFv, etc.).

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

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.

Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus.

PubMed

Langeveld, J P; Brennan, F R; Martínez-Torrecuadrada, J L; Jones, T D; Boshuizen, R S; Vela, C; Casal, J I; Kamstrup, S; Dalsgaard, K; Meloen, R H; Bendig, M M; Hamilton, W D

2001-06-14

A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.

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).

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.

Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

PubMed Central

Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

2011-01-01

Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

Generation of 2A-linked multicistronic cassettes by recombinant PCR.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector. This protocol describes the use of recombinant polymerase chain reaction (PCR) to connect multiple 2A-linked protein sequences. The final construct is subcloned into an expression vector.

Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library.

PubMed

Krumpe, Lauren R H; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2007-10-05

Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

Recombinant Peptides as Biomarkers for Metastatic Breast Cancer Response

DTIC Science & Technology

2007-10-01

could be specific to breast cancer tumor models has just been concluded. In vivo biopanning wsa conducted with a T7 phage -based random peptide library...peptides selected from phage -displayed libraries. 15. SUBJECT TERMS Breast cancer, phage display, molecular imaging, personalized medicine 16...recombinant peptides from phage -displayed peptide libraries can be selected that bind to receptors activated in response to therapy. These peptides in turn

Filamentous bacteriophage as a novel therapeutic tool for Alzheimer's disease treatment.

PubMed

Solomon, Beka

2008-10-01

Antibodies towards the N-terminal region of the amyloid-beta peptide (AbetaP) bind to Abeta fibrils, leading to their disaggregation. We developed an immunization procedure using filamentous phages displaying the only four amino acids EFRH encompassing amino acids 3-6 of the 42 residues of AbetaP, found to be the main regulatory site for Abeta formation. Phages displaying EFRH epitope are effective in eliciting humoral response against AbetaP which, in turn, relieves amyloid burden in brains of amyloid-beta protein precursor transgenic mice, improving their ability to perform cognitive tasks. In order to overcome the low permeability of the blood brain barrier for targeting 'anti-aggregating' monoclonal antibodies (mAbs) to Abeta plaques in the brain, we applied antibody engineering methods to minimize the size of mAbs while maintaining their biological activity. Single-chain antibodies displayed on the surface of filamentous phage showed the ability to enter the central nervous system (CNS). The genetically engineered filamentous bacteriophage proved to be an efficient, nontoxic viral delivery vector to the brain, offering an obvious advantage over other mammalian vectors. The feasibility of these novel strategies for production and targeting of anti-aggregating antibodies against Abeta plaques to disease affected regions in the CNS may have clinical potential for treatment of Alzheimer's disease.

T7 lytic phage-displayed peptide libraries exhibit less sequence bias than M13 filamentous phage-displayed peptide libraries.

PubMed

Krumpe, Lauren R H; Atkinson, Andrew J; Smythers, Gary W; Kandel, Andrea; Schumacher, Kathryn M; McMahon, James B; Makowski, Lee; Mori, Toshiyuki

2006-08-01

We investigated whether the T7 system of phage display could produce peptide libraries of greater diversity than the M13 system of phage display due to the differing processes of lytic and filamentous phage morphogenesis. Using a bioinformatics-assisted computational approach, collections of random peptide sequences obtained from a T7 12-mer library (X(12)) and a T7 7-mer disulfide-constrained library (CX(7)C) were analyzed and compared with peptide populations obtained from New England BioLabs' M13 Ph.D.-12 and Ph.D.-C7C libraries. Based on this analysis, peptide libraries constructed with the T7 system have fewer amino acid biases, increased peptide diversity, and more normal distributions of peptide net charge and hydropathy than the M13 libraries. The greater diversity of T7-displayed libraries provides a potential resource of novel binding peptides for new as well as previously studied molecular targets. To demonstrate their utility, several of the T7-displayed peptide libraries were screened for streptavidin- and neutravidin-binding phage. Novel binding motifs were identified for each protein.

Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.

PubMed

Abes, Saïd; Moulton, Hong M; Clair, Philippe; Prevot, Paul; Youngblood, Derek S; Wu, Rebecca P; Iversen, Patrick L; Lebleu, Bernard

2006-12-01

The efficient and non-toxic nuclear delivery of steric-block oligonucleotides (ON) is a prerequisite for therapeutic strategies involving splice correction or exon skipping. Cationic cell penetrating peptides (CPPs) have given rise to much interest for the intracellular delivery of biomolecules, but their efficiency in promoting cytoplasmic or nuclear delivery of oligonucleotides has been hampered by endocytic sequestration and subsequent degradation of most internalized material in endocytic compartments. In the present study, we compared the splice correction activity of three different CPPs conjugated to PMO(705), a steric-block ON targeted against the mutated splicing site of human beta-globin pre-mRNA in the HeLa pLuc705 splice correction model. In contrast to Tat48-60 (Tat) and oligoarginine (R(9)F(2)) PMO(705) conjugates, the 6-aminohexanoic-spaced oligoarginine (R-Ahx-R)(4)-PMO(705) conjugate was able to promote an efficient splice correction in the absence of endosomolytic agents. Our mechanistic investigations about its uptake mechanisms lead to the conclusion that these three vectors are internalized using the same endocytic route involving proteoglycans, but that the (R-Ahx-R)(4)-PMO(705) conjugate has the unique ability to escape from lysosomial fate and to access to the nuclear compartment. This vector, which has displays an extremely low cytotoxicity, the ability to function without chloroquine adjunction and in the presence of serum proteins. It thus offers a promising lead for the development of vectors able to enhance the delivery of therapeutic steric-block ON in clinically relevant models.

Pharmacologic Effects in vivo in Brain by Vector-Mediated Peptide Drug Delivery

NASA Astrophysics Data System (ADS)

Bickel, Ulrich; Yoshikawa, Takayoshi; Landaw, Elliot M.; Faull, Kym F.; Pardridge, William M.

1993-04-01

Pharmacologic effects in brain caused by systemic administration of neuropeptides are prevented by poor transport of the peptide through the brain vascular endothelium, which comprises the blood-brain barrier in vivo. In the present study, successful application of a chimeric peptide approach to enhance drug delivery through the blood-brain barrier for the purpose of achieving a central nervous system pharmacologic effect is described. The chimeric peptide was formed by linkage of a potent vasoactive intestinal peptide (VIP) analogue, which had been monobiotinylated, to a drug transport vector. The vector consisted of a covalent conjugate of avidin and the OX26 monoclonal antibody to the transferrin receptor. Owing to the high concentration of transferrin receptors on brain capillary endothelia, OX26 targets brain and undergoes receptor-mediated transcytosis through the blood-brain barrier. Systemic infusion of low doses (12 μg/kg) of the VIP chimeric peptide in rats resulted in an in vivo central nervous system pharmacologic effect: a 65% increase in cerebral blood flow. Biotinylated VIP analogue without the brain transport vector was ineffective.

Phage display selection of peptides that target calcium-binding proteins.

PubMed

Vetter, Stefan W

2013-01-01

Phage display allows to rapidly identify peptide sequences with binding affinity towards target proteins, for example, calcium-binding proteins (CBPs). Phage technology allows screening of 10(9) or more independent peptide sequences and can identify CBP binding peptides within 2 weeks. Adjusting of screening conditions allows selecting CBPs binding peptides that are either calcium-dependent or independent. Obtained peptide sequences can be used to identify CBP target proteins based on sequence homology or to quickly obtain peptide-based CBP inhibitors to modulate CBP-target interactions. The protocol described here uses a commercially available phage display library, in which random 12-mer peptides are displayed on filamentous M13 phages. The library was screened against the calcium-binding protein S100B.

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

PubMed

Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

2016-01-01

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

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

M Honda; R Wang; W Kong

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

Different Vaccine Vectors Delivering the Same Antigen Elicit CD8+ T Cell Responses with Distinct Clonotype and Epitope Specificity

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

Honda, M.; Robinson, H.; Wang, R.

Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternativemore » vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.« less

Exploring the role of peptides in polymer-based gene delivery.

PubMed

Sun, Yanping; Yang, Zhen; Wang, Chunxi; Yang, Tianzhi; Cai, Cuifang; Zhao, Xiaoyun; Yang, Li; Ding, Pingtian

2017-09-15

Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors. Polymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides' roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. 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.

Comparison of phage pVIII and KLH as vector in inducing the production of cytokines in C57BL/6J mice.

PubMed

Su, Quan-Ping; Wen, De-Zhong; Yang, Qiong; Zhang, Yan-Hui; Liu, Chong; Wang, Li

2007-01-22

We have demonstrated that phage display Candida albicans (C. albicans) LKVIRK epitope was protective in systemically infected C57BL/6J mice. The different development from precursor Ths, Th1 or Th2, will result in a protective or nonprotective immune response. To compare the types of cytokines induced by biologically and chemically synthesized vectors, C57BL/6J mice were immunized with hybrid phage displaying the epitope of LKVIRK and by synthesized peptide epitope LKVIRKNIVKKMIE conjugated through cysteine to keyhole limpet haemocyanin (KLH). The production of cytokines in spleens of immunized mice and in splenocytes culture supernatants stimulated by homologous immunogen in vitro was studied by RT-PCR and quantitative sandwich ELISA. The results showed that, compared to Tris-EDTA buffer (TE, 1 mM Tris, 0.1 mM EDTA, pH 8.0) injected mice, the expressions of Th1 type cytokine IFN-gamma, IL-2 and IL-12 were increased in hybrid phage, KLH-C, and wild phage immunized mice, and there were no differences between mice immunized with hybrid phage and KLH-C. While the expression of Th2 type cytokine IL-10 was similar in all mice, IL-4 was not detected. We obtained the same results in mRNA and protein level. These findings indicated that as carriers, phage and KLH were similar in inducing the Th1 type cytokines expression. Comparing to peptide synthesis couple with a carrier protein for injection, phage may be an inexpensive and simple route to the production of effective vaccines.

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.

Beyond helper phage: Using "helper cells" to select peptide affinity ligands

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

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

Beyond helper phage: Using "helper cells" to select peptide affinity ligands

DOE PAGES

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin; ...

2016-09-14

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.).

PubMed

Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

2017-01-01

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens.

Screening, Expression, Purification and Functional Characterization of Novel Antimicrobial Peptide Genes from Hermetia illucens (L.)

PubMed Central

Elhag, Osama; Zhou, Dingzhong; Song, Qi; Soomro, Abdul Aziz; Cai, Minmin; Zheng, Longyu; Yu, Ziniu; Zhang, Jibin

2017-01-01

Antimicrobial peptides from a wide spectrum of insects possess potent microbicidal properties against microbial-related diseases. In this study, seven new gene fragments of three types of antimicrobial peptides were obtained from Hermetia illucens (L), and were named cecropinZ1, sarcotoxin1, sarcotoxin (2a), sarcotoxin (2b), sarcotoxin3, stomoxynZH1, and stomoxynZH1(a). Among these genes, a 189-basepair gene (stomoxynZH1) was cloned into the pET32a expression vector and expressed in the Escherichia coli as a fusion protein with thioredoxin. Results show that Trx-stomoxynZH1 exhibits diverse inhibitory activity on various pathogens, including Gram-positive bacterium Staphylococcus aureus, Gram-negative bacterium Escherichia coli, fungus Rhizoctonia solani Khün (rice)-10, and fungus Sclerotinia sclerotiorum (Lib.) de Bary-14. The minimum inhibitory concentration of Trx-stomoxynZH1 is higher against Gram-positive bacteria than against Gram-negative bacteria but similar between the fungal strains. These results indicate that H. illucens (L.) could provide a rich source for the discovery of novel antimicrobial peptides. Importantly, stomoxynZH1 displays a potential benefit in controlling antibiotic-resistant pathogens. PMID:28056070

Peptide Transduction-Based Therapies for Prostate Cancer

DTIC Science & Technology

2004-06-01

using an M13 peptide phage display library. Initial screening of the library for transduction of tumors in vivo has identified peptides able to...marker conjugates may have to be tested. (Months 6-12, Year 1) Progress: These experiments have been initiated. Task 4. An M13 peptide phage display ... phage 12 amino acid control peptide display library (New England Biolabs, Beverly, MA ) was used. Briefly, One nude mouse bearing a human tumor line

Protein Transduction Based Therapies for Breast Cancer

DTIC Science & Technology

2006-07-01

we also have developed a method for screening for tissue-targeted transduction peptides using an M13 peptide phage display library. Using this...Instead the focus was on the ability to identify a tumor specific peptide. Task 4. An M13 peptide phage display library will be used for...cancespecific tumor lines by screening a peptide phage display library both in cell culture as well as in nude micebearing xenografts. Initial results in

Analysis of the role of tripeptidyl peptidase II in MHC class I antigen presentation in vivo1

PubMed Central

Kawahara, Masahiro; York, Ian A.; Hearn, Arron; Farfan, Diego; Rock, Kenneth L.

2015-01-01

Previous experiments using enzyme inhibitors and RNAi in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I antigen presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared to wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits antigen presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus (LCMV). The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild type and TPPII-deficient mice. These data indicate while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several antigens in vivo. PMID:19841172

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector.

PubMed

Mustapa, M Firouz Mohd; Bell, Paul C; Hurley, Christopher A; Nicol, Alastair; Guénin, Erwann; Sarkar, Supti; Writer, Michele J; Barker, Susie E; Wong, John B; Pilkington-Miksa, Michael A; Papahadjopoulos-Sternberg, Brigitte; Shamlou, Parviz Ayazi; Hailes, Helen C; Hart, Stephen L; Zicha, Daniel; Tabor, Alethea B

2007-11-13

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

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.

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.

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.

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

Webb-Robertson, Bobbie-Jo M.

Accurate identification of peptides is a current challenge in mass spectrometry (MS) based proteomics. The standard approach uses a search routine to compare tandem mass spectra to a database of peptides associated with the target organism. These database search routines yield multiple metrics associated with the quality of the mapping of the experimental spectrum to the theoretical spectrum of a peptide. The structure of these results make separating correct from false identifications difficult and has created a false identification problem. Statistical confidence scores are an approach to battle this false positive problem that has led to significant improvements in peptidemore » identification. We have shown that machine learning, specifically support vector machine (SVM), is an effective approach to separating true peptide identifications from false ones. The SVM-based peptide statistical scoring method transforms a peptide into a vector representation based on database search metrics to train and validate the SVM. In practice, following the database search routine, a peptides is denoted in its vector representation and the SVM generates a single statistical score that is then used to classify presence or absence in the sample« less

Isolation and characterization of anti-SEB peptides using magnetic sorting and bacterial peptide display library technology

NASA Astrophysics Data System (ADS)

Pennington, Joseph M.; Kogot, Joshua M.; Sarkes, Deborah A.; Pellegrino, Paul M.; Stratis-Cullum, Dimitra N.

2012-06-01

Peptide display libraries offer an alternative method to existing antibody development methods enabling rapid isolation of highly stable reagents for detection of new and emerging biological threats. Bacterial display libraries are used to isolate new peptide reagents within 1 week, which is simpler and timelier than using competing display library technology based on phage or yeast. Using magnetic sorting methods, we have isolated peptide reagents with high affinity and specificity to staphylococcal enterotoxin B (SEB), a suspected food pathogen. Flow cytometry methods were used for on-cell characterization and the binding affinity (Kd) of this new peptide reagent was determined to be 56 nm with minimal cross-reactivity to other proteins. These results demonstrated that magnetic sorting for new reagents using bacterial display libraries is a rapid and effective method and has the potential for current and new and emerging food pathogen targets.

Recent advances in genetic modification of adenovirus vectors for cancer treatment.

PubMed

Yamamoto, Yuki; Nagasato, Masaki; Yoshida, Teruhiko; Aoki, Kazunori

2017-05-01

Adenoviruses are widely used to deliver genes to a variety of cell types and have been used in a number of clinical trials for gene therapy and oncolytic virotherapy. However, several concerns must be addressed for the clinical use of adenovirus vectors. Selective delivery of a therapeutic gene by adenovirus vectors to target cancer is precluded by the widespread distribution of the primary cellular receptors. The systemic administration of adenoviruses results in hepatic tropism independent of the primary receptors. Adenoviruses induce strong innate and acquired immunity in vivo. Furthermore, several modifications to these vectors are necessary to enhance their oncolytic activity and ensure patient safety. As such, the adenovirus genome has been engineered to overcome these problems. The first part of the present review outlines recent progress in the genetic modification of adenovirus vectors for cancer treatment. In addition, several groups have recently developed cancer-targeting adenovirus vectors by using libraries that display random peptides on a fiber knob. Pancreatic cancer-targeting sequences have been isolated, and these oncolytic vectors have been shown by our group to be associated with a higher gene transduction efficiency and more potent oncolytic activity in cell lines, murine models, and surgical specimens of pancreatic cancer. In the second part of this review, we explain that combining cancer-targeting strategies can be a promising approach to increase the clinical usefulness of oncolytic adenovirus vectors. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Novel DOTA-based prochelator for divalent peptide vectorization: synthesis of dimeric bombesin analogues for multimodality tumor imaging and therapy.

PubMed

Abiraj, Keelara; Jaccard, Hugues; Kretzschmar, Martin; Helm, Lothar; Maecke, Helmut R

2008-07-28

Dimeric peptidic vectors, obtained by the divalent grafting of bombesin analogues on a newly synthesized DOTA-based prochelator, showed improved qualities as tumor targeted imaging probes in comparison to their monomeric analogues.

Development and evaluation of an airplane electronic display format aligned with the inertial velocity vector

NASA Technical Reports Server (NTRS)

Steinmetz, G. G.

1986-01-01

The development of an electronic primary flight display format aligned with the aircraft velocity vector, a simulation evaluation comparing this format with an electronic attitude-aligned primary flight display format, and a flight evaluation of the velocity-vector-aligned display format are described. Earlier tests in turbulent conditions with the electronic attitude-aligned display format had exhibited unsteadiness. A primary objective of aligning the display format with the velocity vector was to take advantage of a velocity-vector control-wheel steering system to provide steadiness of display during turbulent conditions. Better situational awareness under crosswind conditions was also achieved. The evaluation task was a curved, descending approach with turbulent and crosswind conditions. Primary flight display formats contained computer-drawn perspective runway images and flight-path angle information. The flight tests were conducted aboard the NASA Transport Systems Research Vehicle (TSRV). Comparative results of the simulation and flight tests were principally obtained from subjective commentary. Overall, the pilots preferred the display format aligned with the velocity vector.

Verification of 2A peptide cleavage.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. It is now possible to express multiple proteins from a single open reading frame (ORF) using 2A peptide-linked multicistronic vectors. These small sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. The easiest and most effective way to assess 2A cleavage is to perform transient transfection of 293T cells (human embryonic kidney cells) followed by western blot analysis, as described in this protocol. 293T cells are easy to grow and can be efficiently transfected with a variety of vectors. Cleavage can be assessed by detection with antibodies against the target proteins or anti-2A serum.

Development of a Recombinant Multifunctional Biomacromolecule for Targeted Gene Transfer to Prostate Cancer Cells.

PubMed

Hatefi, Arash; Karjoo, Zahra; Nomani, Alireza

2017-09-11

The objective of this study was to genetically engineer a fully functional single chain fusion peptide composed of motifs from diverse biological and synthetic origins that can perform multiple tasks including DNA condensation, cell targeting, cell transfection, particle shielding from immune system and effective gene transfer to prostate tumors. To achieve the objective, a single chain biomacromolecule (vector) consisted of four repeatative units of histone H2A peptide, fusogenic peptide GALA, short elastin-like peptide, and PC-3 cell targeting peptide was designed. To examine the functionality of each motif in the vector sequence, it was characterized in terms of size and zeta potential by Zetasizer, PC-3 cell targeting and transfection by flowcytometry, IgG induction by immunogenicity assay, and PC-3 tumor transfection by quantitative live animal imaging. Overall, the results of this study showed the possibility of using genetic engineering techniques to program various functionalities into one single chain vector and create a multifunctional nonimmunogenic biomacromolecule for targeted gene transfer to prostate cancer cells. This proof-of-concept study is a significant step forward toward creating a library of vectors for targeted gene transfer to any cancer cell type at both in vitro and in vivo levels.

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.

Current state of a dual behaviour of antimicrobial peptides-Therapeutic agents and promising delivery vectors.

PubMed

Piotrowska, Urszula; Sobczak, Marcin; Oledzka, Ewa

2017-12-01

Micro-organism resistance is an important challenge in modern medicine due to the global uncontrolled use of antibiotics. Natural and synthetic antimicrobial peptides (AMPs) symbolize a new family of antibiotics, which have stimulated research and clinical interest as new therapeutic options for infections. They represent one of the most promising antimicrobial substances, due to their broad spectrum of biological activity, against bacteria, fungi, protozoa, viruses, yeast and even tumour cells. Besides, being antimicrobial, AMPs have been shown to bind and neutralize bacterial endotoxins, as well as possess immunomodulatory, anti-inflammatory, wound-healing, angiogenic and antitumour properties. In contrast to conventional antibiotics, which have very defined and specific molecular targets, host cationic peptides show varying, complex and very rapid mechanisms of actions that make it difficult to form an effective antimicrobial defence. Importantly, AMPs display their antimicrobial activity at micromolar concentrations or less. To do this, many peptide-based drugs are commercially available for the treatment of numerous diseases, such as hepatitis C, myeloma, skin infections and diabetes. Herein, we present an overview of the general mechanism of AMPs action, along with recent developments regarding carriers of AMPs and their potential applications in medical fields. © 2017 John Wiley & Sons A/S.

Drug delivery vectors based on filamentous bacteriophages and phage-mimetic nanoparticles.

PubMed

Ju, Zhigang; Sun, Wei

2017-11-01

With the development of nanomedicine, a mass of nanocarriers have been exploited and utilized for targeted drug delivery, including liposomes, polymers, nanoparticles, viruses, and stem cells. Due to huge surface bearing capacity and flexible genetic engineering property, filamentous bacteriophage and phage-mimetic nanoparticles are attracting more and more attentions. As a rod-like bio-nanofiber without tropism to mammalian cells, filamentous phage can be easily loaded with drugs and directly delivered to the lesion location. In particular, chemical drugs can be conjugated on phage surface by chemical modification, and gene drugs can also be inserted into the genome of phage by recombinant DNA technology. Meanwhile, specific peptides/proteins displayed on the phage surface are able to conjugate with nanoparticles which will endow them specific-targeting and huge drug-loading capacity. Additionally, phage peptides/proteins can directly self-assemble into phage-mimetic nanoparticles which may be applied for self-navigating drug delivery nanovehicles. In this review, we summarize the production of phage particles, the identification of targeting peptides, and the recent applications of filamentous bacteriophages as well as their protein/peptide for targeting drug delivery in vitro and in vivo. The improvement of our understanding of filamentous bacteriophage and phage-mimetic nanoparticles will supply new tools for biotechnological approaches.

Validation of SplitVectors Encoding for Quantitative Visualization of Large-Magnitude-Range Vector Fields

PubMed Central

Zhao, Henan; Bryant, Garnett W.; Griffin, Wesley; Terrill, Judith E.; Chen, Jian

2017-01-01

We designed and evaluated SplitVectors, a new vector field display approach to help scientists perform new discrimination tasks on large-magnitude-range scientific data shown in three-dimensional (3D) visualization environments. SplitVectors uses scientific notation to display vector magnitude, thus improving legibility. We present an empirical study comparing the SplitVectors approach with three other approaches - direct linear representation, logarithmic, and text display commonly used in scientific visualizations. Twenty participants performed three domain analysis tasks: reading numerical values (a discrimination task), finding the ratio between values (a discrimination task), and finding the larger of two vectors (a pattern detection task). Participants used both mono and stereo conditions. Our results suggest the following: (1) SplitVectors improve accuracy by about 10 times compared to linear mapping and by four times to logarithmic in discrimination tasks; (2) SplitVectors have no significant differences from the textual display approach, but reduce cluttering in the scene; (3) SplitVectors and textual display are less sensitive to data scale than linear and logarithmic approaches; (4) using logarithmic can be problematic as participants' confidence was as high as directly reading from the textual display, but their accuracy was poor; and (5) Stereoscopy improved performance, especially in more challenging discrimination tasks. PMID:28113469

Validation of SplitVectors Encoding for Quantitative Visualization of Large-Magnitude-Range Vector Fields.

PubMed

Henan Zhao; Bryant, Garnett W; Griffin, Wesley; Terrill, Judith E; Jian Chen

2017-06-01

We designed and evaluated SplitVectors, a new vector field display approach to help scientists perform new discrimination tasks on large-magnitude-range scientific data shown in three-dimensional (3D) visualization environments. SplitVectors uses scientific notation to display vector magnitude, thus improving legibility. We present an empirical study comparing the SplitVectors approach with three other approaches - direct linear representation, logarithmic, and text display commonly used in scientific visualizations. Twenty participants performed three domain analysis tasks: reading numerical values (a discrimination task), finding the ratio between values (a discrimination task), and finding the larger of two vectors (a pattern detection task). Participants used both mono and stereo conditions. Our results suggest the following: (1) SplitVectors improve accuracy by about 10 times compared to linear mapping and by four times to logarithmic in discrimination tasks; (2) SplitVectors have no significant differences from the textual display approach, but reduce cluttering in the scene; (3) SplitVectors and textual display are less sensitive to data scale than linear and logarithmic approaches; (4) using logarithmic can be problematic as participants' confidence was as high as directly reading from the textual display, but their accuracy was poor; and (5) Stereoscopy improved performance, especially in more challenging discrimination tasks.

Advances in the T7 phage display system (Review).

PubMed

Deng, Xiangying; Wang, Li; You, Xiaolong; Dai, Pei; Zeng, Yanhua

2018-01-01

The present review describes the advantages and updated applications of the T7 phage display system in bioscience and medical science. Current phage display systems are based on various bacteriophage vectors, including M13, T7, T4 and f1. Of these, the M13 phage display is the most frequently used, however, the present review highlights the advantages of the T7 system. As a phage display platform, M13 contains single‑stranded DNA, while the T7 phage consists of double‑stranded DNA, which exhibits increased stability and is less prone to mutation during replication. Additional characteristics of the T7 phage include the following: The T7 phage does not depend on a protein secretion pathway in the lytic cycle; expressed peptides and proteins are usually located on the C‑terminal region of capsid protein gp10B, which avoids problems associated with steric hindrance; and T7 phage particles exhibit high stability under various extreme conditions, including high temperature and low pH, which facilitates effective high‑throughput affinity elutriation. Recent applications of the T7 phage display system have been instrumental in uncovering mechanisms of molecular interaction, particularly in the fields of antigen discovery, vaccine development, protein interaction, and cancer diagnosis and treatment.

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.

A human trial of HSV mediated gene transfer for the treatment of chronic pain

PubMed Central

Wolfe, Darren; Mata, Marina; Fink, David J.

2009-01-01

Gene transfer to the dorsal root ganglion using replication defective herpes simplex virus (HSV)-based vectors reduces pain related behaviors in rodent models of inflammatory pain, neuropathic pain, and pain caused by cancer in bone. HSV vectors engineered to produce inhibitory neurotransmitters including the delta opioid agonist peptide enkephalin, the mu opioid agonist peptide endomorphin-2 and glutamic acid decarboxylase (GAD) to effect the release of gamma amino butyric acid (GABA) act to inhibit nociceptive neurotransmission at the first synapse between primary nociceptive and second-order neuron in the dorsal horn of spinal cord. HSV vectors engineered to release anti-inflammatory peptides including interleukin (IL)-4, IL-10 and the p55 soluble tumor necrosis factor α (TNFα) receptor reduce neuroimmune activation in the spinal dorsal horn. The path leading from preclinical animal studies to the ongoing phase 1 human trial of the enkephalin-producing vector in patients with pain from cancer, and plans for an efficacy trial with an opioid producing vector in inflammatory pain and an efficacy trial with a GAD producing vector in diabetic neuropathic pain are outlined. PMID:19242524

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

Dendrimer D5 is a vector for peptide transport to brain cells.

PubMed

Sarantseva, S V; Bolshakova, O I; Timoshenko, S I; Kolobov, A A; Schwarzman, A L

2011-02-01

Dendrimers are a new class of nonviral vectors for gene or drug transport. Dendrimer capacity to penetrate through the blood-brain barrier remaines little studied. Biotinylated polylysine dendrimer D5, similarly to human growth hormone biotinylated fragment covalently bound to D5 dendrimer, penetrates through the blood-brain barrier and accumulates in Drosophila brain after injection into the abdomen. Hence, D5 dendrimer can serve as a vector for peptide transport to brain cells.

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

A Real-Time Phase Vector Display for EEG Monitoring

NASA Technical Reports Server (NTRS)

Finger, Herbert J.; Anliker, James E.; Rimmer, Tamara

1973-01-01

A real-time, computer-based, phase vector display system has been developed which will output a vector whose phase is equal to the delay between a trigger and the peak of a function which is quasi-coherent with respect to the trigger. The system also contains a sliding averager which enables the operator to average successive trials before calculating the phase vector. Data collection, averaging and display generation are performed on a LINC-8 computer. Output displays appear on several X-Y CRT display units and on a kymograph camera/oscilloscope unit which is used to generate photographs of time-varying phase vectors or contourograms of time-varying averages of input functions.

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.

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

Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier.

PubMed

Molino, Yves; David, Marion; Varini, Karine; Jabès, Françoise; Gaudin, Nicolas; Fortoul, Aude; Bakloul, Karima; Masse, Maxime; Bernard, Anne; Drobecq, Lucile; Lécorché, Pascaline; Temsamani, Jamal; Jacquot, Guillaume; Khrestchatisky, Michel

2017-05-01

The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals ( ldlr -/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. © FASEB.

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.

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein.

PubMed

Li, Yifeng

2012-02-01

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount. Copyright © 2011 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.

Dual targeting of gene delivery by genetic modification of adenovirus serotype 5 fibers and cell-selective transcriptional control.

PubMed

Work, L M; Ritchie, N; Nicklin, S A; Reynolds, P N; Baker, A H

2004-08-01

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins.

PubMed

Velappan, Nileena; Fisher, Hugh E; Pesavento, Emanuele; Chasteen, Leslie; D'Angelo, Sara; Kiss, Csaba; Longmire, Michelle; Pavlik, Peter; Bradbury, Andrew R M

2010-03-01

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.

Epitope selection from an uncensored peptide library displayed on avian leukosis virus.

PubMed

Khare, Pranay D; Rosales, Ana G; Bailey, Kent R; Russell, Stephen J; Federspiel, Mark J

2003-10-25

Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10(6) different members was generated from a plasmid library of approximately 5 x 10(6) diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries.

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.

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.

Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

PubMed

Readman, John Benedict; Dickson, George; Coldham, Nick G

2017-06-01

The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-bla CTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying bla CTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

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.

Next-generation ELISA diagnostic assay for Chagas Disease based on the combination of short peptidic epitopes

PubMed Central

Volcovich, Romina; Altcheh, Jaime; Bracamonte, Estefanía; Marco, Jorge D.; Nielsen, Morten; Buscaglia, Carlos A.

2017-01-01

Chagas Disease, caused by the protozoan Trypanosoma cruzi, is a major health and economic problem in Latin America for which no vaccine or appropriate drugs for large-scale public health interventions are yet available. Accurate diagnosis is essential for the early identification and follow up of vector-borne cases and to prevent transmission of the disease by way of blood transfusions and organ transplantation. Diagnosis is routinely performed using serological methods, some of which require the production of parasite lysates, parasite antigenic fractions or purified recombinant antigens. Although available serological tests give satisfactory results, the production of reliable reagents remains laborious and expensive. Short peptides spanning linear B-cell epitopes have proven ideal serodiagnostic reagents in a wide range of diseases. Recently, we have conducted a large-scale screening of T. cruzi linear B-cell epitopes using high-density peptide chips, leading to the identification of several hundred novel sequence signatures associated to chronic Chagas Disease. Here, we performed a serological assessment of 27 selected epitopes and of their use in a novel multipeptide-based diagnostic method. A combination of 7 of these peptides were finally evaluated in ELISA format against a panel of 199 sera samples (Chagas-positive and negative, including sera from Leishmaniasis-positive subjects). The multipeptide formulation displayed a high diagnostic performance, with a sensitivity of 96.3% and a specificity of 99.15%. Therefore, the use of synthetic peptides as diagnostic tools are an attractive alternative in Chagas’ disease diagnosis. PMID:28991925

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.

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.

Construction of genetically engineered M13K07 helper phage for simultaneous phage display of gold binding peptide 1 and nuclear matrix protein 22 ScFv antibody.

PubMed

Fatemi, Farnaz; Amini, Seyed Mohammad; Kharrazi, Sharmin; Rasaee, Mohammad Javad; Mazlomi, Mohammad Ali; Asadi-Ghalehni, Majid; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2017-11-01

The most common techniques of antibody phage display are based on the use of M13 filamentous bacteriophages. This study introduces a new genetically engineered M13K07 helper phage displaying multiple copies of a known gold binding peptide on p8 coat proteins. The recombinant helper phages were used to rescue a phagemid vector encoding the p3 coat protein fused to the nuclear matrix protein 22 (NMP22) ScFv antibody. Transmission electron microscopy (TEM), UV-vis absorbance spectroscopy, and field emission scanning electron microscopy (FE-SEM) with energy dispersive X-ray spectroscopy (EDX) analysis revealed that the expression of gold binding peptide 1 (GBP1) on major coat protein p8 significantly enhances the gold-binding affinity of M13 phages. The recombinant bacteriophages at concentrations above 5×10 4 pfu/ml red-shifted the UV-vis absorbance spectra of gold nanoparticles (AuNPs); however, the surface plasmon resonance of gold nanoparticles was not changed by the wild type bacteriophages at concentrations up to 10 12 pfu/ml. The phage ELISA assay demonstrated the high affinity binding of bifunctional bacteriophages to NMP22 antigen at concentrations of 10 5 and 10 6 pfu/ml. Thus, the p3 end of the bifunctional bacteriophages would be able to bind to specific target antigen, while the AuNPs were assembled along the coat of virus for signal generation. Our results indicated that the complex of antigen-bacteriophages lead to UV-vis spectral changes of AuNPs and NMP22 antigen in concentration range of 10-80μg/ml can be detected by bifunctional bacteriophages at concentration of 10 4 pfu/ml. The ability of bifunctional bacteriophages to bind to antigen and generate signal at the same time, makes this approach applicable for identifying different antigens in immunoassay techniques. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioreducible Fluorinated Peptide Dendrimers Capable of Circumventing Various Physiological Barriers for Highly Efficient and Safe Gene Delivery.

PubMed

Cai, Xiaojun; Jin, Rongrong; Wang, Jiali; Yue, Dong; Jiang, Qian; Wu, Yao; Gu, Zhongwei

2016-03-09

Polymeric vectors have shown great promise in the development of safe and efficient gene delivery systems; however, only a few have been developed in clinical settings due to poor transport across multiple physiological barriers. To address this issue and promote clinical translocation of polymeric vectors, a new type of polymeric vector, bioreducible fluorinated peptide dendrimers (BFPDs), was designed and synthesized by reversible cross-linking of fluorinated low generation peptide dendrimers. Through masterly integration all of the features of reversible cross-linking, fluorination, and polyhedral oligomeric silsesquioxane (POSS) core-based peptide dendrimers, this novel vector exhibited lots of unique features, including (i) inactive surface to resist protein interactions; (ii) virus-mimicking surface topography to augment cellular uptake; (iii) fluorination-mediated efficient cellular uptake, endosome escape, cytoplasm trafficking, and nuclear entry, and (iv) disulfide-cleavage-mediated polyplex disassembly and DNA release that allows efficient DNA transcription. Noteworthy, all of these features are functionally important and can synergistically facilitate DNA transport from solution to the nucleus. As a consequences, BFPDs showed excellent gene transfection efficiency in several cell lines (∼95% in HEK293 cells) and superior biocompatibility compared with polyethylenimine (PEI). Meanwhile BFPDs provided excellent serum resistance in gene delivery. More importantly, BFPDs offer considerable in vivo gene transfection efficiency (in muscular tissues and in HepG2 tumor xenografts), which was approximately 77-fold higher than that of PEI in luciferase activity. These results suggest bioreducible fluorinated peptide dendrimers are a new class of highly efficient and safe gene delivery vectors and should be used in clinical settings.

Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors

PubMed Central

Maschauer, Simone; Einsiedel, Jürgen; Reich, Dominik; Hübner, Harald; Gmeiner, Peter; Wester, Hans-Jürgen; Prante, Olaf; Notni, Johannes

2017-01-01

Neurotensin receptor 1 (NTS1) is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET). The metabolically-stabilized neurotensin (NT) derivative peptide Nlys8-Lys9-Pro10-Tyr11-Tle12-Leu13-OH was elongated at the N-terminus with 6-azido norleucine and coupled with the 1,4,7-triazacyclononane-1,4,7-tris[(2-carboxyethyl)methylenephosphinic acid] (TRAP) chelator TRAP(alkyne)3 in order to synthesize a NT trimer with subnanomolar affinity and high stability. The 68Ga-labeled peptide [68Ga]Ga-TRAP(NT4)3 was characterized in vitro using the NTS1-expressing human colorectal adenocarcinoma cell line HT29. It displayed fast and high internalization rates of >90%, but also fast efflux rates of 50% over 15 min. In vivo, [68Ga]Ga-TRAP(NT4)3 showed moderate HT29 tumor uptake values of 1.7 %ID/g at 60 min post-injection (p.i.), but also high uptake and retention in the kidneys and liver. A comparison of data for trimer/monomer pairs of NT ligands and other targeting vectors (peptides and peptoids targeting integrins αvβ3, α5β1, and αvβ6, the PSMA-ligand DUPA (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), and nitroimidazoles targeting hypoxia) revealed that multimers always exhibit higher target affinities and tumor uptake, but not necessarily improved tumor-to-tissue ratios. Thus, although in vitro data are not suitable for prediction of in vivo performance, multimers are potentially superior to monomers, particularly for applications where high tumor accumulation is crucial. PMID:28287433

A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages

USDA-ARS?s Scientific Manuscript database

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral vectored subunits, and synthetic peptides, most of which suffer from poor immunogenicity and are subject to degradation. For these reasons, efficient delivery systems and potent immunost...

No recent adaptive selection on the apyrase of Mediterranean Phlebotomus: implications for using salivary peptides to vaccinate against canine leishmaniasis.

PubMed

Mahamdallie, Shazia S; Ready, Paul D

2012-04-01

Vaccine development is informed by a knowledge of genetic variation among antigen alleles, especially the distribution of positive and balancing selection in populations and species. A combined approach using population genetic and phylogenetic methods to detect selective signatures can therefore be informative for identifying vaccine candidates. Parasitic Leishmania species cause the disease leishmaniasis in humans and mammalian reservoir hosts after inoculation by female phlebotomine sandflies. Like other arthropod vectors of disease agents, sandflies use salivary peptides to counteract host haemostatic and immunomodulatory responses during bloodfeeding, and these peptides are vaccine candidates because they can protect against Leishmania infection. We detected no contemporary adaptive selection on one salivary peptide, apyrase, in 20 populations of Phlebotomus ariasi, a European vector of Leishmania infantum. Maximum likelihood branch models on a gene phylogeny showed apyrase to be a single copy in P. ariasi but an ancient duplication event associated with temporary positive selection was observed in its sister group, which contains most Mediterranean vectors of L. infantum. The absence of contemporary adaptive selection on the apyrase of P. ariasi may result from this sandfly's opportunistic feeding behaviour. Our study illustrates how the molecular population genetics of arthropods can help investigate the potential of salivary peptides for disease control and for understanding geographical variation in vector competence.

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.

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.

Highly efficient, nonpeptidic oligoguanidinium vectors that selectively internalize into mitochondria.

PubMed

Fernández-Carneado, Jimena; Van Gool, Michiel; Martos, Vera; Castel, Susanna; Prados, Pilar; de Mendoza, Javier; Giralt, Ernest

2005-01-26

Oligoguanidinium-based cell delivery systems have gained broad interest in the drug delivery field since one decade ago. Thus, arginine-containing peptides as Tat or Antp, oligoarginine peptides, and derived peptoids have been described as shuttles for delivering nonpermeant drugs inside cancer cells. Herein we report a new family of tetraguanidinium cell penetrating vectors efficiently internalized in human tumor cells. Their high internalization, studied by confocal microscopy and flow cytometry, as well as their specific accumulation in mitochondria makes these new vectors likely vehicles for the targeted delivery of anticancer drugs to mitochondria.

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

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.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

PubMed

Jacquot, Guillaume; Lécorché, Pascaline; Malcor, Jean-Daniel; Laurencin, Mathieu; Smirnova, Maria; Varini, Karine; Malicet, Cédric; Gassiot, Fanny; Abouzid, Karima; Faucon, Aude; David, Marion; Gaudin, Nicolas; Masse, Maxime; Ferracci, Géraldine; Dive, Vincent; Cisternino, Salvatore; Khrestchatisky, Michel

2016-12-05

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC] c DPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC] c ) which specifically binds hLDLR with a K D of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC] c ), which showed the highest K D value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 10 6 s -1. M -1 range, and off-rates varying from the low 10 -2 s -1 to the 10 -1 s -1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"] c ), showing a K D of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3 H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

In vivo gene delivery and expression by bacteriophage lambda vectors.

PubMed

Lankes, H A; Zanghi, C N; Santos, K; Capella, C; Duke, C M P; Dewhurst, S

2007-05-01

Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo. Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer. Unmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up -- at least in part -- by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer. These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

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.

Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area

PubMed Central

Elanga Ndille, Emmanuel; Doucoure, Souleymane; Poinsignon, Anne; Mouchet, François; Cornelie, Sylvie; D’Ortenzio, Eric; DeHecq, Jean Sébastien; Remoue, Franck

2016-01-01

Background Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes. Methodology/Principal findings Specific human IgG response to the Nterm-34kDa peptide was assessed from 102 individuals living in urban area of Saint-Denis at La Reunion Island, Indian Ocean, before and after the implementation of vector control against Aedes mosquitoes. IgG response decreased after 2 weeks (P < 0.0001), and remained low for 4 weeks post-intervention (P = 0.0002). The specific IgG decrease was associated with the decline of Aedes mosquito density, as estimated by entomological parameters and closely correlated to vector control implementation and was not associated with the use of individual protection, daily commuting outside of the house, sex and age. Our findings indicate a probable short-term decrease of human exposure to Aedes bites just after vector control implementation. Conclusion/Significance Results provided in the present study indicate that IgG Ab response to Aedes aegypti Nterm-34kDa salivary peptide could be a relevant short-time indicator for evaluating the efficacy of vector control interventions against Aedes species. PMID:27906987

Human IgG Antibody Response to Aedes Nterm-34kDa Salivary Peptide, an Epidemiological Tool to Assess Vector Control in Chikungunya and Dengue Transmission Area.

PubMed

Elanga Ndille, Emmanuel; Doucoure, Souleymane; Poinsignon, Anne; Mouchet, François; Cornelie, Sylvie; D'Ortenzio, Eric; DeHecq, Jean Sébastien; Remoue, Franck

2016-12-01

Arboviral diseases are an important public health concerns. Vector control remains the sole strategy to fight against these diseases. Because of the important limits of methods currently used to assess human exposure to Aedes mosquito bites, much effort is being devoted to develop new indicators. Recent studies have reported that human antibody (Ab) responses to Aedes aegypti Nterm-34kDa salivary peptide represent a promising biomarker tool to evaluate the human-Aedes contact. The present study aims investigate whether such biomarker could be used for assessing the efficacy of vector control against Aedes. Specific human IgG response to the Nterm-34kDa peptide was assessed from 102 individuals living in urban area of Saint-Denis at La Reunion Island, Indian Ocean, before and after the implementation of vector control against Aedes mosquitoes. IgG response decreased after 2 weeks (P < 0.0001), and remained low for 4 weeks post-intervention (P = 0.0002). The specific IgG decrease was associated with the decline of Aedes mosquito density, as estimated by entomological parameters and closely correlated to vector control implementation and was not associated with the use of individual protection, daily commuting outside of the house, sex and age. Our findings indicate a probable short-term decrease of human exposure to Aedes bites just after vector control implementation. Results provided in the present study indicate that IgG Ab response to Aedes aegypti Nterm-34kDa salivary peptide could be a relevant short-time indicator for evaluating the efficacy of vector control interventions against Aedes species.

AAVP displaying octreotide for ligand-directed therapeutic transgene delivery in neuroendocrine tumors of the pancreas.

PubMed

Smith, Tracey L; Yuan, Ziqiang; Cardó-Vila, Marina; Sanchez Claros, Carmen; Adem, Asha; Cui, Min-Hui; Branch, Craig A; Gelovani, Juri G; Libutti, Steven K; Sidman, Richard L; Pasqualini, Renata; Arap, Wadih

2016-03-01

Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.

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.

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.

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...

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.

Peptides, polypeptides and peptide-polymer hybrids as nucleic acid carriers.

PubMed

Ahmed, Marya

2017-10-24

Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.

3D hydrophobic moment vectors as a tool to characterize the surface polarity of amphiphilic peptides.

PubMed

Reißer, Sabine; Strandberg, Erik; Steinbrecher, Thomas; Ulrich, Anne S

2014-06-03

The interaction of membranes with peptides and proteins is largely determined by their amphiphilic character. Hydrophobic moments of helical segments are commonly derived from their two-dimensional helical wheel projections, and the same is true for β-sheets. However, to the best of our knowledge, there exists no method to describe structures in three dimensions or molecules with irregular shape. Here, we define the hydrophobic moment of a molecule as a vector in three dimensions by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule, e.g., partial unfolding, bending, and side-chain torsion angles. Extended all-atom molecular dynamics simulations are then used to calculate the equilibrium hydrophobic moments for two antimicrobial peptides, gramicidin S and PGLa, under different conditions. We show that their effective hydrophobic moment vectors reflect the distribution of polar and nonpolar patches on the molecular surface and the calculated electrostatic surface potential. A comparison of simulations in solution and in lipid membranes shows how the peptides undergo internal conformational rearrangement upon binding to the bilayer surface. A good correlation with solid-state NMR data indicates that the hydrophobic moment vector can be used to predict the membrane binding geometry of peptides. This method is available as a web application on http://www.ibg.kit.edu/HM/. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

PubMed Central

Liu, Kehai; Wang, Xiaoyu; Fan, Wei; Zhu, Qing; Yang, Jingya; Gao, Jing; Gao, Shen

2012-01-01

Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy. PMID:22412301

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.

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.

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.

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.

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.

Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries.

PubMed

Zanconato, Stefano; Minervini, Giovanni; Poli, Irene; De Lucrezia, Davide

2011-01-01

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.

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.

Human Antibody Responses to the Anopheles Salivary gSG6-P1 Peptide: A Novel Tool for Evaluating the Efficacy of ITNs in Malaria Vector Control

PubMed Central

Drame, Papa Makhtar; Poinsignon, Anne; Besnard, Patrick; Cornelie, Sylvie; Le Mire, Jacques; Toto, Jean-Claude; Foumane, Vincent; Dos-Santos, Maria Adelaide; Sembène, Mbacké; Fortes, Filomeno; Simondon, Francois; Carnevale, Pierre; Remoue, Franck

2010-01-01

To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of Anopheles gambiae salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to Anopheles salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to Anopheles. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact. PMID:21179476

Human antibody responses to the Anopheles salivary gSG6-P1 peptide: a novel tool for evaluating the efficacy of ITNs in malaria vector control.

PubMed

Drame, Papa Makhtar; Poinsignon, Anne; Besnard, Patrick; Cornelie, Sylvie; Le Mire, Jacques; Toto, Jean-Claude; Foumane, Vincent; Dos-Santos, Maria Adelaide; Sembène, Mbacké; Fortes, Filomeno; Simondon, Francois; Carnevale, Pierre; Remoue, Franck

2010-12-14

To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of Anopheles gambiae salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to Anopheles salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to Anopheles. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact.

The establishment of Saccharomyces boulardii surface display system using a single expression vector.

PubMed

Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

2014-03-01

In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.

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

Bio-inorganic synthesis of ZnO powders using recombinant His-tagged ZnO binding peptide as a promoter.

PubMed

Song, Lei; Liu, Yingying; Zhang, Zhifang; Wang, Xi; Chen, Jinchun

2010-10-01

Inorganic-binding peptides termed as genetically engineered polypeptides for inorganics (GEPIs), are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. Recent advances in nanotechnology and molecular biology allow the engineering of these peptides with specific affinity to inorganics, often used as molecular linkers or assemblers, to facilitate materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here we report a novel biosynthetic ZnO binding protein and its application in promoting bio-inorganic materials synthesis. In brief, the gene encoding a ZnO binding peptide(ZBP) was genetically fused with His(6)-tag and GST-tag using E.coli expression vector pET-28a (+) and pGEX-4T-3. The recombinant protein GST-His-ZBP was expressed, purified with Ni-NTA system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. Affinity adsorption test demonstrated that the fusion protein had a specific avidity for ZnO nanoparticles (NPs). Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)(2) sol. X-ray diffraction (XRD) analysis on the final products after calcining the precursor powders showed that hexagonal wurtzite ZnO crystals were obtained. Our work suggested a novel approach to the application about the organic-inorganic interactions.

Adapter-directed display: a modular design for shuttling display on phage surfaces.

PubMed

Wang, Kevin Caili; Wang, Xinwei; Zhong, Pingyu; Luo, Peter Peizhi

2010-02-05

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems. Copyright 2009 Elsevier Ltd. All rights reserved.

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.

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.

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

Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

PubMed

White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

2008-12-01

Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

Facilitating protein solubility by use of peptide extensions

DOEpatents

Freimuth, Paul I; Zhang, Yian-Biao; Howitt, Jason

2013-09-17

Expression vectors for expression of a protein or polypeptide of interest as a fusion product composed of the protein or polypeptide of interest fused at one terminus to a solubility enhancing peptide extension are provided. Sequences encoding the peptide extensions are provided. The invention further comprises antibodies which bind specifically to one or more of the solubility enhancing peptide extensions.

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.

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

Blood-brain barrier shuttle peptides enhance AAV transduction in the brain after systemic administration.

PubMed

Zhang, Xintao; He, Ting; Chai, Zheng; Samulski, R Jude; Li, Chengwen

2018-09-01

The adeno-associated virus (AAV) vector has been used in preclinical and clinical trials of gene therapy for central nervous system (CNS) diseases. One of the biggest challenges of effectively delivering AAV to the brain is to surmount the blood-brain barrier (BBB). Herein, we identified several potential BBB shuttle peptides that significantly enhanced AAV8 transduction in the brain after a systemic administration, the best of which was the THR peptide. The enhancement of AAV8 brain transduction by THR is dose-dependent, and neurons are the primary THR targets. Mechanism studies revealed that THR directly bound to the AAV8 virion, increasing its ability to cross the endothelial cell barrier. Further experiments showed that binding of THR to the AAV virion did not interfere with AAV8 infection biology, and that THR competitively blocked transferrin from binding to AAV8. Taken together, our results demonstrate, for the first time, that BBB shuttle peptides are able to directly interact with AAV and increase the ability of the AAV vectors to cross the BBB for transduction enhancement in the brain. These results will shed important light on the potential applications of BBB shuttle peptides for enhancing brain transduction with systemic administration of AAV vectors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Establishment of MicroRNA delivery system by PP7 bacteriophage-like particles carrying cell-penetrating peptide.

PubMed

Sun, Yanli; Sun, Yanhua; Zhao, Ronglan

2017-08-01

MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low in vivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system. Here we showed that PP7 VLPs carrying a CPP penetrated hepatoma SK-HEP-1 cells and delivered the pre-microRNA-23b, which was processed into a mature product within 24 h; a concentration of 10 nM was sufficient for the inhibition of hepatoma cell migration via the downregulation of liver-intestine cadherin expression. Furthermore, PP7 VLPs carrying a CPP and a pre-microRNA were not infectious, replicative, or cytotoxic. Therefore, recombinant PP7 VLPs can be used for simultaneous and targeted delivery of both microRNAs and peptides because of their ability to package specific exogenous RNA using the pac site and to display peptides. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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.

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.

T7 lytic phage-displayed peptide libraries: construction and diversity characterization.

PubMed

Krumpe, Lauren R H; Mori, Toshiyuki

2014-01-01

In this chapter, we describe the construction of T7 bacteriophage (phage)-displayed peptide libraries and the diversity analyses of random amino acid sequences obtained from the libraries. We used commercially available reagents, Novagen's T7Select system, to construct the libraries. Using a combination of biotinylated extension primer and streptavidin-coupled magnetic beads, we were able to prepare library DNA without applying gel purification, resulting in extremely high ligation efficiencies. Further, we describe the use of bioinformatics tools to characterize library diversity. Amino acid frequency and positional amino acid diversity and hydropathy are estimated using the REceptor LIgand Contacts website http://relic.bio.anl.gov. Peptide net charge analysis and peptide hydropathy analysis are conducted using the Genetics Computer Group Wisconsin Package computational tools. A comprehensive collection of the estimated number of recombinants and titers of T7 phage-displayed peptide libraries constructed in our lab is included.

A split ubiquitin system to reveal topology and released peptides of membrane proteins.

PubMed

Li, Qiu-Ping; Wang, Shuai; Gou, Jin-Ying

2017-09-02

Membrane proteins define biological functions of membranes in cells. Extracellular peptides of transmembrane proteins receive signals from pathogens or environments, and are the major targets of drug developments. Despite of their essential roles, membrane proteins remain elusive in topological studies due to technique difficulties in their expressions and purifications. First, the target gene is cloned into a destination vector to fuse with C terminal ubiquitin at the N or C terminus. Then, Cub vector with target gene and Nub WT or Nub G vectors are transformed into AP4 or AP5 yeast cells, respectively. After mating, the diploid cells are dipped onto selection medium to check the growth. Topology of the target protein is determined according to Table 1. We present a split ubiquitin topology (SUT) analysis system to study the topology and truncation peptide of membrane proteins in a simple yeast experiment. In the SUT system, transcription activator (TA) fused with a nucleo-cytoplasmic protein shows strong auto-activation with both positive and negative control vectors. TA fused with the cytoplasmic end of membrane proteins activates reporter genes only with positive control vector with a wild type N terminal ubiquitin (Nub WT ). However, TA fused with the extracellular termini of membrane proteins can't activate reporter genes even with Nub WT . Interestingly,TA fused with the released peptide of a membrane protein shows autoactivation in the SUT system. The SUT system is a simple and fast experimental procedure complementary to computational predictions and large scale proteomic techniques. The preliminary data from SUT are valuable for pathogen recognitions and new drug developments.

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.

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

PubMed

White, K; Büning, H; Kritz, A; Janicki, H; McVey, J; Perabo, L; Murphy, G; Odenthal, M; Work, L M; Hallek, M; Nicklin, S A; Baker, A H

2008-03-01

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection.

PubMed

Bayer, Wibke; Tenbusch, Matthias; Lietz, Ruth; Johrden, Lena; Schimmer, Simone; Uberla, Klaus; Dittmer, Ulf; Wildner, Oliver

2010-02-01

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

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

Methods and materials for deconstruction of biomass for biofuels production

DOEpatents

Schoeniger, Joseph S; Hadi, Masood Zia

2015-05-05

The present invention relates to nucleic acids, peptides, vectors, cells, and plants useful in the production of biofuels. In certain embodiments, the invention relates to nucleic acid sequences and peptides from extremophile organisms, such as SSO1949 and Ce1A, that are useful for hydrolyzing plant cell wall materials. In further embodiments, the invention relates to modified versions of such sequences that have been optimized for production in one or both of monocot and dicot plants. In other embodiments, the invention provides for targeting peptide production or activity to a certain location within the cell or organism, such as the apoplast. In further embodiments, the invention relates to transformed cells or plants. In additional embodiments, the invention relates to methods of producing biofuel utilizing such nucleic acids, peptides, targeting sequences, vectors, cells, and/or plants.

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

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.

Expression and purification of moricin CM4 and human β-defensins 4 in Escherichia coli using a new technology.

PubMed

Shen, Yang; Ai, Hong-Xin; Song, Ren; Liang, Zhen-Ning; Li, Jian-Feng; Zhang, Shuang-Quan

2010-10-20

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human β-defensins 4 (HβD4) in the Escherichia coli. The CM4 and HβD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HβD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide. Copyright © 2010 Elsevier GmbH. All rights reserved.

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.

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.

Developing Novel Therapeutics Targeting Undifferentiated and Castration-Resistant Prostate Cancer Stem Cells

DTIC Science & Technology

2016-10-01

identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to identify novel PCSC-targeting chemicals. In the past...display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides ; and 2) To perform unbiased drug library screening to...Goals of the Project (SOW): Aim 1: To perform phage display library (PDL) screening in PSA-/lo PCa cells to identify PCSC- specific homing peptides

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

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.

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

Gene-carried hepatoma targeting complex induced high gene transfection efficiency with low toxicity and significant antitumor activity.

PubMed

Zhao, Qing-Qing; Hu, Yu-Lan; Zhou, Yang; Li, Ni; Han, Min; Tang, Gu-Ping; Qiu, Feng; Tabata, Yasuhiko; Gao, Jian-Qing

2012-01-01

The success of gene transfection is largely dependent on the development of a vehicle or vector that can efficiently deliver a gene to cells with minimal toxicity. A liver cancer-targeted specific peptide (FQHPSF sequence) was successfully synthesized and linked with chitosan-linked polyethylenimine (CP) to form a new targeted gene delivery vector called CPT (CP/peptide). The structure of CPT was confirmed by (1)H nuclear magnetic resonance spectroscopy and ultraviolet spectrophotometry. The particle size of CPT/ DNA complexes was measured using laser diffraction spectrometry and the cytotoxicity of the copolymer was evaluated by methylthiazol tetrazolium method. The transfection efficiency evaluation of the CP copolymer was performed using luciferase activity assay. Cellular internalization of the CP/DNA complex was observed under confocal laser scanning microscopy. The targeting specificity of the polymer coupled to peptide was measured by competitive inhibition transfection study. The liver targeting specificity of the CPT copolymer in vivo was demonstrated by combining the copolymer with a therapeutic gene, interleukin-12, and assessed by its abilities in suppressing the growth of ascites tumor in mouse model. The results showed that the liver cancer-targeted specific peptide was successfully synthesized and linked with CP to form a new targeted gene delivery vector called CPT. The composition of CPT was confirmed and the vector showed low cytotoxicity and strong targeting specificity to liver tumors in vitro. The in vivo study results showed that interleukin-12 delivered by the new gene vector CPT/DNA significantly enhanced the antitumor effect on ascites tumor-bearing imprinting control region mice as compared with polyethylenimine (25 kDa), CP, and other controls, which further demonstrate the targeting specificity of the new synthesized polymer. The synthesized CPT copolymer was proven to be an effective liver cancer-targeted vector for therapeutic gene delivery, which could be a potential candidate for targeted cancer gene therapy.

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.

Orally Delivered Scorpion Antimicrobial Peptides Exhibit Activity against Pea Aphid (Acyrthosiphon pisum) and Its Bacterial Symbionts.

PubMed

Luna-Ramirez, Karen; Skaljac, Marisa; Grotmann, Jens; Kirfel, Phillipp; Vilcinskas, Andreas

2017-08-24

Aphids are severe agricultural pests that damage crops by feeding on phloem sap and vectoring plant pathogens. Chemical insecticides provide an important aphid control strategy, but alternative and sustainable control measures are required to avoid rapidly emerging resistance, environmental contamination, and the risk to humans and beneficial organisms. Aphids are dependent on bacterial symbionts, which enable them to survive on phloem sap lacking essential nutrients, as well as conferring environmental stress tolerance and resistance to parasites. The evolution of aphids has been accompanied by the loss of many immunity-related genes, such as those encoding antibacterial peptides, which are prevalent in other insects, probably because any harm to the bacterial symbionts would inevitably affect the aphids themselves. This suggests that antimicrobial peptides (AMPs) could replace or at least complement conventional insecticides for aphid control. We fed the pea aphids ( Acyrthosiphon pisum ) with AMPs from the venom glands of scorpions. The AMPs reduced aphid survival, delayed their reproduction, displayed in vitro activity against aphid bacterial symbionts, and reduced the number of symbionts in vivo. Remarkably, we found that some of the scorpion AMPs compromised the aphid bacteriome, a specialized organ that harbours bacterial symbionts. Our data suggest that scorpion AMPs holds the potential to be developed as bio-insecticides, and are promising candidates for the engineering of aphid-resistant crops.

Orally Delivered Scorpion Antimicrobial Peptides Exhibit Activity against Pea Aphid (Acyrthosiphon pisum) and Its Bacterial Symbionts

PubMed Central

Luna-Ramirez, Karen; Skaljac, Marisa; Grotmann, Jens; Kirfel, Phillipp; Vilcinskas, Andreas

2017-01-01

Aphids are severe agricultural pests that damage crops by feeding on phloem sap and vectoring plant pathogens. Chemical insecticides provide an important aphid control strategy, but alternative and sustainable control measures are required to avoid rapidly emerging resistance, environmental contamination, and the risk to humans and beneficial organisms. Aphids are dependent on bacterial symbionts, which enable them to survive on phloem sap lacking essential nutrients, as well as conferring environmental stress tolerance and resistance to parasites. The evolution of aphids has been accompanied by the loss of many immunity-related genes, such as those encoding antibacterial peptides, which are prevalent in other insects, probably because any harm to the bacterial symbionts would inevitably affect the aphids themselves. This suggests that antimicrobial peptides (AMPs) could replace or at least complement conventional insecticides for aphid control. We fed the pea aphids (Acyrthosiphon pisum) with AMPs from the venom glands of scorpions. The AMPs reduced aphid survival, delayed their reproduction, displayed in vitro activity against aphid bacterial symbionts, and reduced the number of symbionts in vivo. Remarkably, we found that some of the scorpion AMPs compromised the aphid bacteriome, a specialized organ that harbours bacterial symbionts. Our data suggest that scorpion AMPs holds the potential to be developed as bio-insecticides, and are promising candidates for the engineering of aphid-resistant crops. PMID:28837113

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.

Antigenic properties of HCMV peptides displayed by filamentous bacteriophages vs. synthetic peptides.

PubMed

Ulivieri, Cristina; Citro, Alessandra; Ivaldi, Federico; Mascolo, Dina; Ghittoni, Raffaella; Fanigliulo, Daniela; Manca, Fabrizio; Baldari, Cosima Tatiana; Li Pira, Giuseppina; Del Pozzo, Giovanna

2008-08-15

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.

Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

PubMed Central

Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

2014-01-01

Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

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.

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.

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

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.

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.

Phage display peptide libraries: deviations from randomness and correctives

PubMed Central

Ryvkin, Arie; Ashkenazy, Haim; Weiss-Ottolenghi, Yael; Piller, Chen; Pupko, Tal; Gershoni, Jonathan M

2018-01-01

Abstract Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications. PMID:29420788

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.

Lipopolysaccharide interactions of C-terminal peptides from human thrombin.

PubMed

Singh, Shalini; Kalle, Martina; Papareddy, Praveen; Schmidtchen, Artur; Malmsten, Martin

2013-05-13

Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

Prediction of cell penetrating peptides by support vector machines.

PubMed

Sanders, William S; Johnston, C Ian; Bridges, Susan M; Burgess, Shane C; Willeford, Kenneth O

2011-07-01

Cell penetrating peptides (CPPs) are those peptides that can transverse cell membranes to enter cells. Once inside the cell, different CPPs can localize to different cellular components and perform different roles. Some generate pore-forming complexes resulting in the destruction of cells while others localize to various organelles. Use of machine learning methods to predict potential new CPPs will enable more rapid screening for applications such as drug delivery. We have investigated the influence of the composition of training datasets on the ability to classify peptides as cell penetrating using support vector machines (SVMs). We identified 111 known CPPs and 34 known non-penetrating peptides from the literature and commercial vendors and used several approaches to build training data sets for the classifiers. Features were calculated from the datasets using a set of basic biochemical properties combined with features from the literature determined to be relevant in the prediction of CPPs. Our results using different training datasets confirm the importance of a balanced training set with approximately equal number of positive and negative examples. The SVM based classifiers have greater classification accuracy than previously reported methods for the prediction of CPPs, and because they use primary biochemical properties of the peptides as features, these classifiers provide insight into the properties needed for cell-penetration. To confirm our SVM classifications, a subset of peptides classified as either penetrating or non-penetrating was selected for synthesis and experimental validation. Of the synthesized peptides predicted to be CPPs, 100% of these peptides were shown to be penetrating.

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli.

PubMed

Cao, Wei; Zhou, Yuxun; Ma, Yushu; Luo, Qingping; Wei, Dongzhi

2005-04-01

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.

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

Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

PubMed Central

2011-01-01

Background Several naturally occurring cationic antimicrobial peptides (CAPs), including bovine lactoferricin (LfcinB), display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS) on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS) on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS. PMID:21453492

Chemical and genetic wrappers for improved phage and RNA display.

PubMed

Lamboy, Jorge A; Tam, Phillip Y; Lee, Lucie S; Jackson, Pilgrim J; Avrantinis, Sara K; Lee, Hye J; Corn, Robert M; Weiss, Gregory A

2008-11-24

An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.

Predictive Features of a Cockpit Traffic Display: A Workload Assessment

NASA Technical Reports Server (NTRS)

Wickens, Christopher D.; Morphew, Ephimia

1997-01-01

Eighteen pilots flew a series of traffic avoidance maneuvers in an experiment designed to assess the support offered and workload imposed by different levels of traffic display information in a free flight simulation. Three display prototypes were compared which differed in traffic information provided. A BASELINE (BL) display provided current and (2nd order) predicted information regarding ownship and current information of an intruder aircraft, represented on lateral and vertical displays in a coplanar suite. An INTRUDER PREDICTOR (IP) display, augmented the baseline display by providing lateral and vertical prediction of the intruder aircraft. A THREAT VECTOR (TV) display added to the IP display a vector that indicates the direction from ownship to the intruder at the predicted point of closest contact (POCC). The length of the vector corresponds to the radius of the protected zone, and the distance of the intersection of the vector with ownship predictor, corresponds to the time available till POCC or loss of separation. Pilots time shared the traffic avoidance task with a secondary task requiring them to monitor the top of the display for faint targets. This task simulated the visual demands of out-of-cockpit scanning, and hence was used to estimate the head-down time required by the different display formats. The results revealed that both display augmentations improved performance (safety) as assessed by predicted and actual loss of separation (i.e., penetration of the protected zone). Both enhancements also reduced workload, as assessed by the NASA TLX scale. The intruder predictor display produced these benefits with no substantial impact on the qualitative nature of the avoidance maneuvers that were selected. The threat vector produced the safety benefits by inducing a greater degree of (effective) lateral maneuvering, thus partially offsetting the benefits of reduced workload. The three displays did not differ in terms of their effect on performance of the monitoring task, used to infer head-down time, nor in the extent of vertical or airspeed maneuvering. The results are discussed in terms of their implications for 19 cognitive engineering design features.

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis.

PubMed

Trentin, Diana; Hall, Heike; Wechsler, Sandra; Hubbell, Jeffrey A

2006-02-21

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

PubMed

Martin, Irene; Dohmen, Christian; Mas-Moruno, Carlos; Troiber, Christina; Kos, Petra; Schaffert, David; Lächelt, Ulrich; Teixidó, Meritxell; Günther, Michael; Kessler, Horst; Giralt, Ernest; Wagner, Ernst

2012-04-28

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer. This journal is © The Royal Society of Chemistry 2012

Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Herranz, Carmen; Gutiérrez, Jorge; Criado, Raquel; Hernández, Pablo E.; Cintas, Luis M.

2009-01-01

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter PGAL1. The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFα1s-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system. PMID:19218405

Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

PubMed

Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

2016-03-15

Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Concurrent display of both α- and β-turns in a model peptide.

PubMed

Srinivas, Deekonda; Vijayadas, Kuruppanthara N; Gonnade, Rajesh; Phalgune, Usha D; Rajamohanan, Pattuparambil R; Sanjayan, Gangadhar J

2011-08-21

This article describes a model peptide that concurrently displays both α- and β-turns, as demonstrated by structural investigations using single crystal X-ray crystallography and solution-state NMR studies. The motif reported herein has the potential for the design of novel conformationally ordered synthetic oligomers with structural architectures distinct from those classically observed.

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.

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

The adsorption of preferential binding peptides to apatite-based materials

PubMed Central

Segvich, Sharon J.; Smith, Hayes C.; Kohn, David H.

2009-01-01

The objective of this work was to identify peptide sequences with high affinity to bone-like mineral (BLM) to provide alternative design methods for functional bone regeneration peptides. Adsorption of preferential binding peptide sequences on four apatite-based substrates [BLM and three sintered apatite disks pressed from powders containing 0% CO32− (HA), 5.6% CO32− (CA5), 10.5% CO32− (CA10)] with varied compositions and morphologies was investigated. A combination of phage display, ELISA, and computational modeling was used to elucidate three 12-mer peptide sequences APWHLSSQYSRT (A), STLPI-PHEFSRE (S), and VTKHLNQISQSY (V), from 243 candidates with preferential adsorption on BLM and HA. Overall, peptides S and V have a significantly higher adsorption to the apatite-based materials in comparison to peptide A (for S vs. A, BLM p = 0.001, CA5 p < 0.001, CA10 p < 0.001, HA p = 0.038; for V vs. A, BLM p = 0.006, CA5 p = 0.033, CA10 p = 0.029). FT-IR analysis displayed carbonate levels in CA5 and CA10 dropped to approximately 1.1–2.2% after sintering, whereas SEM imaging displayed CA5 and CA10 possess distinct morphologies. Adsorption results normalized to surface area indicate that small changes in carbonate percentage at a similar morphological scale did not provide enough carbonate incorporation to show statistical differences in peptide adsorption. Because the identified peptides (S and V) have preferential binding to apatite, their use can now be investigated in bone and dentin tissue engineering, tendon and ligament repair, and enamel formation. PMID:19095299

Representation and display of vector field topology in fluid flow data sets

NASA Technical Reports Server (NTRS)

Helman, James; Hesselink, Lambertus

1989-01-01

The visualization of physical processes in general and of vector fields in particular is discussed. An approach to visualizing flow topology that is based on the physics and mathematics underlying the physical phenomenon is presented. It involves determining critical points in the flow where the velocity vector vanishes. The critical points, connected by principal lines or planes, determine the topology of the flow. The complexity of the data is reduced without sacrificing the quantitative nature of the data set. By reducing the original vector field to a set of critical points and their connections, a representation of the topology of a two-dimensional vector field that is much smaller than the original data set but retains with full precision the information pertinent to the flow topology is obtained. This representation can be displayed as a set of points and tangent curves or as a graph. Analysis (including algorithms), display, interaction, and implementation aspects are discussed.

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

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

PubMed Central

Skamel, Claudia; Aller, Stephen G.; Bopda Waffo, Alain

2014-01-01

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets. PMID:25393763

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.

Heterologous Protein Secretion in Lactobacilli with Modified pSIP Vectors

PubMed Central

Karlskås, Ingrid Lea; Maudal, Kristina; Axelsson, Lars; Rud, Ida; Eijsink, Vincent G. H.; Mathiesen, Geir

2014-01-01

We describe new variants of the modular pSIP-vectors for inducible gene expression and protein secretion in lactobacilli. The basic functionality of the pSIP system was tested in Lactobacillus strains representing 14 species using pSIP411, which harbors the broad-host-range Lactococcus lactis SH71rep replicon and a β-glucuronidase encoding reporter gene. In 10 species, the inducible gene expression system was functional. Based on these results, three pSIP vectors with different signal peptides were modified by replacing their narrow-host-range L. plantarum 256rep replicon with SH71rep and transformed into strains of five different species of Lactobacillus. All recombinant strains secreted the target protein NucA, albeit with varying production levels and secretion efficiencies. The Lp_3050 derived signal peptide generally resulted in the highest levels of secreted NucA. These modified pSIP vectors are useful tools for engineering a wide variety of Lactobacillus species. PMID:24614815

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

Text analysis devices, articles of manufacture, and text analysis methods

DOEpatents

Turner, Alan E; Hetzler, Elizabeth G; Nakamura, Grant C

2015-03-31

Text analysis devices, articles of manufacture, and text analysis methods are described according to some aspects. In one aspect, a text analysis device includes a display configured to depict visible images, and processing circuitry coupled with the display and wherein the processing circuitry is configured to access a first vector of a text item and which comprises a plurality of components, to access a second vector of the text item and which comprises a plurality of components, to weight the components of the first vector providing a plurality of weighted values, to weight the components of the second vector providing a plurality of weighted values, and to combine the weighted values of the first vector with the weighted values of the second vector to provide a third vector.

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.

Routes for Drug Translocation Across the Blood-Brain Barrier: Exploiting Peptides as Delivery Vectors.

PubMed

Kristensen, Mie; Brodin, Birger

2017-09-01

A number of potent drugs for the treatment of brain diseases are available. However, in order for them to reach their target site of action, they must pass the blood-brain barrier (BBB). The capillary endothelium comprises the major barrier of the BBB and allows only passive permeation of some small lipophilic molecules. Brain delivery of the larger biopharmaceuticals, which today includes an increasing number of novel drug entities, is therefore restricted, both due to their molecular size and their hydrophilic nature. Thus, the development of novel drug entities intended for the treatment of brain diseases such as neurodegenerative diseases or brain cancers require a delivery strategy for overcoming the BBB before reaching its final target within the brain. Peptide-based delivery vector is an emerging tool as shuttles for drug delivery across the BBB and one may explore receptor-mediated transcytosis, adsorptive-mediated transcytosis, and the paracellular route. The latter, however, being controversial due to the risk of co-delivery of blood-borne potential harmful substances. On the other hand, a number of studies report on drug delivery across the BBB exploiting receptor-mediated transcytosis and adsorptive-mediated transcytosis, indicating that peptides and peptide vectors may be of use in a central nervous system delivery context. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum.

PubMed

Losurdo, Luca; Quintieri, Laura; Caputo, Leonardo; Gallerani, Raffaele; Mayo, Baltasar; De Leo, Francesca

2013-03-01

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 μg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 μg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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.

Discovery of an artificial peptide agonist to the fibroblast growth factor receptor 1c/βKlotho complex from random peptide T7 phage display.

PubMed

Sakamoto, Kotaro; Kawata, Yayoi; Masuda, Yasushi; Umemoto, Tadashi; Ito, Takashi; Asami, Taiji; Takekawa, Shiro; Ohtaki, Tetsuya; Inooka, Hiroshi

2016-11-04

Fibroblast growth factor receptor-1c (FGFR1c)/βKlotho (KLB) complex is a receptor of fibroblast growth factor 21 (FGF21). Pharmacologically, FGF21 shows anti-obesity and anti-diabetic effects upon peripheral administration. Here, we report the development of an artificial peptide agonist to the FGFR1c/KLB heterodimer complex. The peptide, F91-8A07 (LPGRTCREYPDLWWVRCY), was discovered from random peptide T7 phage display and selectively bound to the FGFR1c/KLB complex, but not to FGFR1c and KLB individually. After subsequent peptide dimerization using a short polyethyleneglycol (PEG) linker, the dimeric F91-8A07 peptide showed higher potent agonist activity than that of FGF21 in cultured primary human adipocytes. Moreover, the dimeric peptide led to an expression of the early growth response protein-1 (Egr-1) mRNA in vivo, which is a target gene of FGFR1c. To the best of our knowledge, this is the first report of a FGFR1c/KLB complex-selective artificial peptide agonist. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Decreasing the configurational entropy and the hydrophobicity of EBV-derived peptide 11389 increased its antigenicity, immunogenicity and its ability of inducing IL-6.

PubMed

Urquiza, Mauricio; Guevara, Tatiana; Rodriguez, Cristina; Melo-Cardenas, Johanna; Vanegas, Magnolia; Patarroyo, Manuel E

2012-06-01

Peptide 11389 from CD21-binding region of EBV-gp350/220 protein binds to PBMCs inducing IL-6 expression and inhibiting EBV-binding to PBMCs. In addition, anti-peptide 11389 antibodies recognize EBV-infected cells and inhibit both EBV infection and IL-6 production in PBMCs. We have postulated that native structure stabilization of peptide 11389 sequence can increase its biological activity. The strategy was to modify its sequence to restrict the number of structures that peptide 11389 could acquire in solution (decreasing peptide's configurational entropy) and to weaken the non-relevant intermolecular interactions (decreasing its hydrophobicity), preserving CD21-interacting residues and structure as displayed in the native protein. Thirteen analog peptides were designed and synthesized; most of them were monomers containing an intra-chain disulfide bridge. Analog peptides 34058, 34060, 34061, 34296, 34298, 34299 and 34300 inhibited EBV invasion of PBMCs. Peptides 34059, 34060, 34295 and 34297 induced IL-6 levels in PBMCs (EC50=3.4, 3.3, 0.5, 0.5 μM, respectively) at higher potency than peptide 11389 (EC50=5.8 μM). Peptides 34057, 34059, 34060, 34301 and 34302 interacted with anti-EBV antibodies with affinities from 3 to 50 times higher than peptide 11389. Most of analog peptides were highly immunogenic and elicited antibodies that cross-react with EBV. In conclusion, we have designed peptides displaying higher biological activity than peptide 11389.

Biomedical Applications of Organometal-Peptide Conjugates

NASA Astrophysics Data System (ADS)

Metzler-Nolte, Nils

Peptides are well suited as targeting vectors for the directed delivery of metal-based drugs or probes for biomedical investigations. This chapter describes synthetic strategies for the preparation of conjugates of medically interesting peptides with covalently bound metal complexes. Peptides that were used include neuropeptides (enkephalin, neuropeptide Y, neurotensin), uptake peptides (TAT and poly-Arg), and intracellular localization sequences. To these peptides, a whole variety of transition metal complexes has been attached in recent years by solid-phase peptide synthesis (SPPS) techniques. The metal complex can be attached to the peptide on the resin as part of the SPPS scheme. Alternatively, the metal complex may be attached to the peptide as a postsynthetic modification. Advantages as well as disadvantages for either strategy are discussed. Biomedical applications include radiopharmaceutical applications, anticancer and antibacterial activity, metal-peptide conjugates as targeted CO-releasing molecules, and metal-peptide conjugates in biosensor applications.

Expression of amyloid-beta 1-40 and 1-42 peptides in Capsicum annum var. angulosum for oral immunization.

PubMed

Szabó, Beáta; Hori, Koichi; Nakajima, Akiko; Sasagawa, Noboru; Watanabe, Yuichiro; Ishiura, Shoichi

2004-08-01

Alzheimer's disease (AD), the leading cause of dementia in the elderly population, still remains without an effective treatment. The accumulation and deposition of the amyloid-beta peptide (Abeta) in the brain is thought to be a key event in the pathogenesis of AD. Recently, a novel exciting technology has been investigated to combat AD: new immunotherapeutic approaches have been described that are based on vaccination with the Abeta peptide itself, and this has been shown to induce functionally beneficial anti-Abeta antibody responses in different transgenic animal models of AD. Here we report the high level expression of GFP-Abeta1-40 and 1-42 peptides in Capsicum annum var. angulosum (green pepper) using a new tomato mosaic tobamovirus-based hybrid replication vector. After preinoculation of Nicotiana benthamiana plants with the in vitro transcript of the vector, the isolated virions were used to inoculate green pepper, which accumulated the GFPAbeta1-40 or 1-42 fusion proteins to a level of 100 microg/g of leaves 7 days after inoculation. These results make it possible to test whether oral immunization by feeding plant samples could stimulate antibody production against Abeta peptides.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

PubMed

Bogacheva, Mariia; Egorova, Anna; Slita, Anna; Maretina, Marianna; Baranov, Vladislav; Kiselev, Anton

2017-11-01

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway

PubMed Central

Marsden, Catherine J.; Lord, J. Michael; Roberts, Lynne M.

2003-01-01

Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass. PMID:12734560

Complex formation and vectorization of a phosphorothioate oligonucleotide with an amphipathic leucine- and lysine-rich peptide: study at molecular and cellular levels.

PubMed

Boukhalfa-Heniche, Fatima-Zohra; Hernández, Belén; Gaillard, Stéphane; Coïc, Yves-Marie; Huynh-Dinh, Tam; Lecouvey, Marc; Seksek, Olivier; Ghomi, Mahmoud

2004-04-15

Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C. Copyright 2004 Wiley Periodicals, Inc.

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.

Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

PubMed

Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. 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.

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.

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.

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.

Silica formation with nanofiber morphology via helical display of the silaffin R5 peptide on a filamentous bacteriophage.

PubMed

Song, In-Wong; Park, Hyojung; Park, Jung Han; Kim, Hyunook; Kim, Seong Hun; Yi, Sung; Jaworski, Justyn; Sang, Byoung-In

2017-11-24

Biological systems often generate unique and useful structures, which can have industrial relevance either as direct components or as an inspiration for biomimetic materials. For fabrication of nanoscale silica structures, we explored the use of the silaffin R5 peptide from Cylindrotheca fusiformis expressed on the surface of the fd bacteriophage. By utilizing the biomineralizing peptide component displayed on the bacteriophage surface, we found that low concentrations (0.09 mg/mL of the R5 bacteriophage, below the concentration range used in other studies) could be used to create silica nanofibers. An additional benefit of this approach is the ability of our R5-displaying phage to form silica materials without the need for supplementary components, such as aminopropyl triethoxysilane, that are typically used in such processes. Because this method for silica formation can occur under mild conditions when implementing our R5 displaying phage system, we may provide a relatively simple, economical, and environmentally friendly process for creating silica nanomaterials.

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum.

PubMed

Christiansen, Anders; Kringelum, Jens V; Hansen, Christian S; Bøgh, Katrine L; Sullivan, Eric; Patel, Jigar; Rigby, Neil M; Eiwegger, Thomas; Szépfalusi, Zsolt; de Masi, Federico; Nielsen, Morten; Lund, Ole; Dufva, Martin

2015-08-06

Phage display is a prominent screening technique with a multitude of applications including therapeutic antibody development and mapping of antigen epitopes. In this study, phages were selected based on their interaction with patient serum and exhaustively characterised by high-throughput sequencing. A bioinformatics approach was developed in order to identify peptide motifs of interest based on clustering and contrasting to control samples. Comparison of patient and control samples confirmed a major issue in phage display, namely the selection of unspecific peptides. The potential of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage display by (i) enabling the analysis of complex biological samples, (ii) circumventing the traditional laborious picking and functional testing of individual phage clones and (iii) reducing the number of selection rounds.

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

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

A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide

PubMed Central

Morizono, Kouki; Xie, Yiming; Helguera, Gustavo; Daniels, Tracy R.; Lane, Timothy F.; Penichet, Manuel L.; Chen, Irvin S. Y.

2010-01-01

Background Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin. Methods We inserted the biotin-adaptor-peptide, which was biotinylated by secretory biotin ligase at specific sites, into our targeting envelope proteins, enabling conjugation of the pseudotyped virus with avidin, streptavidin or neutravidin. Results When conjugated with avidin-antibody fusion proteins or the complex of avidin and biotinylated targeting molecules, the vectors could mediate specific transduction to targeted cells recognized by the targeting molecules. When conjugated with streptavidin-coated magnetic beads, transduction by the vectors was targeted to the locations of magnets. Conclusions This targeting vector system can be used for broad applications of targeted gene transduction using biotinylated targeting molecules or targeting molecules fused with avidin. PMID:19455593

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.

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Immunogenic Subviral Particles Displaying Domain III of Dengue 2 Envelope Protein Vectored by Measles Virus

PubMed Central

Harahap-Carrillo, Indira S.; Ceballos-Olvera, Ivonne; Reyes-del Valle, Jorge

2015-01-01

Vaccines against dengue virus (DV) are commercially nonexistent. A subunit vaccination strategy may be of value, especially if a safe viral vector acts as biologically active adjuvant. In this paper, we focus on an immunoglobulin-like, independently folded domain III (DIII) from DV 2 envelope protein (E), which contains epitopes that elicits highly specific neutralizing antibodies. We modified the hepatitis B small surface antigen (HBsAg, S) in order to display DV 2 DIII on a virus-like particle (VLP), thus generating the hybrid antigen DIII-S. Two varieties of measles virus (MV) vectors were developed to express DIII-S. The first expresses the hybrid antigen from an additional transcription unit (ATU) and the second additionally expresses HBsAg from a separate ATU. We found that this second MV vectoring the hybrid VLPs displaying DIII-S on an unmodified HBsAg scaffold were immunogenic in MV-susceptible mice (HuCD46Ge-IFNarko), eliciting robust neutralizing responses (averages) against MV (1:1280 NT90), hepatitis B virus (787 mIU/mL), and DV2 (1:160 NT50) in all of the tested animals. Conversely, the MV vector expressing only DIII-S induced immunity against MV alone. In summary, DV2 neutralizing responses can be generated by displaying E DIII on a scaffold of HBsAg-based VLPs, vectored by MV. PMID:26350592

Quantitative modeling of peptide binding to TAP using support vector machine.

PubMed

Diez-Rivero, Carmen M; Chenlo, Bernardo; Zuluaga, Pilar; Reche, Pedro A

2010-01-01

The transport of peptides to the endoplasmic reticulum by the transporter associated with antigen processing (TAP) is a necessary step towards determining CD8 T cell epitopes. In this work, we have studied the predictive performance of support vector machine models trained on single residue positions and residue combinations drawn from a large dataset consisting of 613 nonamer peptides of known affinity to TAP. Predictive performance of these TAP affinity models was evaluated under 10-fold cross-validation experiments and measured using Pearson's correlation coefficients (R(p)). Our results show that every peptide position (P1-P9) contributes to TAP binding (minimum R(p) of 0.26 +/- 0.11 was achieved by a model trained on the P6 residue), although the largest contributions to binding correspond to the C-terminal end (R(p) = 0.68 +/- 0.06) and the P1 (R(p) = 0.51 +/- 0.09) and P2 (0.57 +/- 0.08) residues of the peptide. Training the models on additional peptide residues generally improved their predictive performance and a maximum correlation (R(p) = 0.89 +/- 0.03) was achieved by a model trained on the full-length sequences or a residue selection consisting of the first 5 N- and last 3 C-terminal residues of the peptides included in the training set. A system for predicting the binding affinity of peptides to TAP using the methods described here is readily available for free public use at http://imed.med.ucm.es/Tools/tapreg/. (c) 2009 Wiley-Liss, Inc.

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.

Rapid Development of New Protein Biosensors Utilizing Peptides Obtained via Phage Display

DTIC Science & Technology

2011-10-01

removal of loosely bound peptide or the viscosity/density change of solutions. ALT sensor operation QCM , CV and EIS measurements validated the formation...manuscript, we demonstrate this process from start to finish to create a new biosensor for the detection of ALT. Figure 6. Sensor operation. A) QCM ...peptides, peptide synthesis with a terminal thiol, QCM in-situ monitoring of peptide immobilization, and sensor detection using electrochemical techniques

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.

ABI domain containing proteins contribute to surface protein display and cell division in Staphylococcus aureus

PubMed Central

Frankel, Matthew B.; Wojcik, Brandon; DeDent, Andrea C.; Missiakas, Dominique M.; Schneewind, Olaf

2012-01-01

Summary The human pathogen Staphyloccocus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harbored transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross walls and in the relative abundance of staphylococci with cross walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. PMID:20923422

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

PubMed

Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

15N backbone dynamics of the S-peptide from ribonuclease A in its free and S-protein bound forms: toward a site-specific analysis of entropy changes upon folding.

PubMed Central

Alexandrescu, A. T.; Rathgeb-Szabo, K.; Rumpel, K.; Jahnke, W.; Schulthess, T.; Kammerer, R. A.

1998-01-01

Backbone 15N relaxation parameters (R1, R2, 1H-15N NOE) have been measured for a 22-residue recombinant variant of the S-peptide in its free and S-protein bound forms. NMR relaxation data were analyzed using the "model-free" approach (Lipari & Szabo, 1982). Order parameters obtained from "model-free" simulations were used to calculate 1H-15N bond vector entropies using a recently described method (Yang & Kay, 1996), in which the form of the probability density function for bond vector fluctuations is derived from a diffusion-in-a-cone motional model. The average change in 1H-15N bond vector entropies for residues T3-S15, which become ordered upon binding of the S-peptide to the S-protein, is -12.6+/-1.4 J/mol.residue.K. 15N relaxation data suggest a gradient of decreasing entropy values moving from the termini toward the center of the free peptide. The difference between the entropies of the terminal and central residues is about -12 J/mol residue K, a value comparable to that of the average entropy change per residue upon complex formation. Similar entropy gradients are evident in NMR relaxation studies of other denatured proteins. Taken together, these observations suggest denatured proteins may contain entropic contributions from non-local interactions. Consequently, calculations that model the entropy of a residue in a denatured protein as that of a residue in a di- or tri-peptide, might over-estimate the magnitude of entropy changes upon folding. PMID:9521116

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.

In vitro digestive stability of complexes between gliadin and synthetic blocking peptides.

PubMed

Hoffmann, Karolina; Carlsson, Nils-Gunnar; Alminger, Marie; Chen, Tingsu; Wold, Agnes; Olsson, Olof; Sandberg, Ann-Sofie

2011-05-01

Celiac disease is caused by an inappropriate immune response to incompletely digested gluten proteins. We investigated whether synthetic peptides with high affinity to wheat gliadin could be selected with a phage display technique and whether complexes between such peptides and gliadin could sustain gastric and pancreatic digestion. Two synthetic peptides, P61 and P64, were selected because of their high affinity to immobilized gliadin. They were allowed to form complexes with gliadin, whereafter the complexes were subjected to in vitro digestion with gastric and pancreatic enzymes. The digestion products were analyzed with Western blot and RP HPLC. The results showed that both peptides formed stable complexes with intact gliadin and that complexes between gliadin and peptide P64 partly resisted gastrointestinal digestion. The two peptides reduced the binding of serum anti-gliadin IgA antibodies by 12%, and 11.5%, respectively, and the binding of anti-gliadin antibodies of the IgG isotype by 13% and 10%. Thus peptides produced by a phage display technique could interact stably with gliadin partly masking epitopes for antibody binding. A combination of peptides of this kind may be used to block gliadin-immune system interactions. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

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.

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR.

PubMed

Monjezi, Razieh; Tan, Sheau Wei; Tey, Beng Ti; Sieo, Chin Chin; Tan, Wen Siang

2013-01-01

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Use of Phage Display technology in development of canine visceral leishmaniasis vaccine using synthetic peptide trapped in sphingomyelin/cholesterol liposomes.

PubMed

Toledo-Machado, Christina Monerat; Bueno, Lilian Lacerda; Menezes-Souza, Daniel; Machado-de-Avila, Ricardo Andrez; Nguyen, Christophe; Granier, Claude; Bartholomeu, Daniella Castanheira; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

2015-02-28

Leishmania parasites can cause visceral or cutaneous disease and are found in subtropical and tropical regions of the Old and New World. The pathology of the infection is determined by both host immune factors and species/strain differences of the parasite. Dogs represent the major reservoir of Leishmania infantum (syn. L. chagasi) and vaccines are considered the most cost-effective control tools for canine disease. Selection of immunodominant peptides was performed by Phage Display to identify sequences recognized by L. infantum naturally infected animals. Sera from Leishmania infected animals were used in the biopanning to selection of specific peptides. Serum samples from T. cruzi infected and healthy animals were used as control. After selection, synthetic peptides were produced in membrane (spot-synthesis) in soluble form and blotting and ELISA were performed for validation of serum reactivity. Selected peptide was formulated with aluminum hydroxide and liposomes and immunization was performed in BALB/c mice. Protection was determined by qPCR after challenge infection with virulent L. infantum. We reported the selection of Peptide 5 through Phage Display technique and demonstrate its ability to promote a state of immunity against L. infantum infection in murine model after immunization using liposomes as vaccine carrier. Our results demonstrate that immunization with Peptide 5 when formulated with aluminum hydroxide and liposomes is immunogenic and elicited significant protection associated with the induction of mixed Th1/Th2 immune response against L. infantum infection. Peptide 5 is a promising vaccine candidate and the findings obtained in the present study encourage canine trials to confirm the effectiveness of a vaccine against CVL.

The tick plasma lectin, Dorin M, is a fibrinogen-related molecule.

PubMed

Rego, Ryan O M; Kovár, Vojtĕch; Kopácek, Petr; Weise, Christoph; Man, Petr; Sauman, Ivo; Grubhoffer, Libor

2006-04-01

A lectin, named Dorin M, previously isolated and characterized from the hemolymph plasma of the soft tick, Ornithodoros moubata, was cloned and sequenced. The immunofluorescence using confocal microscopy revealed that Dorin M is produced in the tick hemocytes. A tryptic cleavage of Dorin M was performed and the resulting peptide fragments were sequenced by Edman degradation and/or mass spectrometry. Two of three internal peptide sequences displayed a significant similarity to the family of fibrinogen-related molecules. Degenerate primers were designed and used for PCR with hemocyte cDNA as a template. The sequence of the whole Dorin M cDNA was completed by the method of RACE. The tissue-specific expression investigated by RT-PCR revealed that Dorin M, in addition to hemocytes, is significantly expressed in salivary glands. The derived amino-acid sequence clearly shows that Dorin M has a fibrinogen-like domain, and exhibited the most significant similarity with tachylectins 5A and 5B from a horseshoe crab, Tachypleus tridentatus. In addition, other protein and binding characteristics suggest that Dorin M is closely related to tachylectins-5. Since these lectins have been reported to function as non-self recognizing molecules, we believe that Dorin M may play a similar role in an innate immunity of the tick and, possibly, also in pathogen transmission by this vector.

A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

PubMed

Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

2012-01-01

Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

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

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

The use of computer-generated color graphic images for transient thermal analysis. [for hypersonic aircraft

NASA Technical Reports Server (NTRS)

Edwards, C. L. W.; Meissner, F. T.; Hall, J. B.

1979-01-01

Color computer graphics techniques were investigated as a means of rapidly scanning and interpreting large sets of transient heating data. The data presented were generated to support the conceptual design of a heat-sink thermal protection system (TPS) for a hypersonic research airplane. Color-coded vector and raster displays of the numerical geometry used in the heating calculations were employed to analyze skin thicknesses and surface temperatures of the heat-sink TPS under a variety of trajectory flight profiles. Both vector and raster displays proved to be effective means for rapidly identifying heat-sink mass concentrations, regions of high heating, and potentially adverse thermal gradients. The color-coded (raster) surface displays are a very efficient means for displaying surface-temperature and heating histories, and thereby the more stringent design requirements can quickly be identified. The related hardware and software developments required to implement both the vector and the raster displays for this application are also discussed.

Structure-activity studies and therapeutic potential of host defense peptides of human thrombin.

PubMed

Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Mörgelin, Matthias; Albiger, Barbara; Malmsten, Martin; Schmidtchen, Artur

2011-06-01

Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs.

PubMed

Santos, Marcus Rebouças; Assao, Viviane Sisdelli; Santos, Fabiana de Almeida Araújo; Salgado, Rafael Locatelli; Carneiro, Ana Paula; Fietto, Juliana Lopes Rangel; Bressan, Gustavo Costa; de Almeida, Márcia Rogéria; Lobato, Zelia Inês Portela; Ueira-Veira, Carlos; Goulart, Luíz Ricardo; Silva-Júnior, Abelardo

2018-07-01

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51-CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.

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.

Combining Chemoselective Ligation with Polyhistidine-Driven Self-Assembly for the Modular Display of Biomolecules on Quantum Dots

PubMed Central

Prasuhn, Duane E.; Blanco-Canosa, Juan B.; Vora, Gary J.; Delehanty, James B.; Susumu, Kimihiro; Mei, Bing C.; Dawson, Philip E.; Medintz, Igor L.

2015-01-01

One of the principle hurdles to wider incorporation of semiconductor quantum dots (QDs) in biology is the lack of facile linkage chemistries to create different types of functional QD-bioconjugates. A two-step modular strategy for the presentation of biomolecules on CdSe/ZnS core/shell QDs is described here which utilizes a chemoselective, aniline-catalyzed hydrazone coupling chemistry to append hexahistidine sequences onto peptides and DNA. This specifically provides them the ability to ratiometrically self-assemble to hydrophilic QDs. The versatility of this labeling approach was highlighted by ligating proteolytic substrate peptides, an oligoarginine cell-penetrating peptide, or a DNA-probe to cognate hexahistidine peptidyl sequences. The modularity allowed subsequently self-assembled QD constructs to engage in different types of targeted bioassays. The self-assembly and photophysical properties of individual QD conjugates were first confirmed by gel electrophoresis and Förster resonance energy transfer analysis. QD-dye-labeled peptide conjugates were then used as biosensors to quantitatively monitor the proteolytic activity of caspase-3 or elastase enzymes from different species. These sensors allowed the determination of the corresponding kinetic parameters, including the Michaelis constant (KM) and the maximum proteolytic activity (Vmax). QDs decorated with cell-penetrating peptides were shown to be successfully internalized by HEK 293T/17 cells, while nanocrystals displaying peptide-DNA conjugates were utilized as fluorescent probes in hybridization microarray assays. This modular approach for displaying peptides or DNA on QDs may be extended to other more complex biomolecules such as proteins or utilized with different types of nanoparticle materials. PMID:20099912

Improvement of the Efficacy of Linear Undecapeptides against Plant-Pathogenic Bacteria by Incorporation of d-Amino Acids ▿

PubMed Central

Güell, Imma; Cabrefiga, Jordi; Badosa, Esther; Ferre, Rafael; Talleda, Montserrat; Bardají, Eduard; Planas, Marta; Feliu, Lidia; Montesinos, Emilio

2011-01-01

A set of 31 undecapeptides, incorporating 1 to 11 d-amino acids and derived from the antimicrobial peptide BP100 (KKLFKKILKYL-NH2), was designed and synthesized. This set was evaluated for inhibition of growth of the plant-pathogenic bacteria Erwinia amylovora, Pseudomonas syringae pv. syringae, and Xanthomonas axonopodis pv. vesicatoria, hemolysis, and protease degradation. Two derivatives were as active as BP100, and 10 peptides displayed improved activity, with the all-d isomer being the most active. Twenty-six peptides were less hemolytic than BP100, and all peptides were more stable against protease degradation. Plant extracts inhibited the activity of BP100 as well as that of the d-isomers. Ten derivatives incorporating one d-amino acid each were tested in an infectivity inhibition assay with the three plant-pathogenic bacteria by using detached pear and pepper leaves and pear fruits. All 10 peptides studied were active against E. amylovora, 6 displayed activity against P. syringae pv. syringae, and 2 displayed activity against X. axonopodis pv. vesicatoria. Peptides BP143 (KKLFKKILKYL-NH2) and BP145 (KKLFKKILKYL-NH2), containing one d-amino acid at positions 4 and 2 (underlined), respectively, were evaluated in whole-plant assays for the control of bacterial blight of pepper and pear and fire blight of pear. Peptide BP143 was as effective as streptomycin in the three pathosystems, was more effective than BP100 against bacterial blight of pepper and pear, and equally effective against fire blight of pear. PMID:21335383

New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation.

PubMed

Landais, Elise; Romagnoli, Pablo A; Corper, Adam L; Shires, John; Altman, John D; Wilson, Ian A; Garcia, K Christopher; Teyton, Luc

2009-12-15

Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4(+)-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A(d)-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

Biological Templating and the Production of Functional Fibers

DTIC Science & Technology

2006-11-01

technique to express designed functional peptides on the virus surface is so-called phage display . It has been widely used to modify the virus surface...and functionality. By using the phage display technique, short peptides containing 2 to 12 random amino acids can be fused into pIII proteins to... M13 filamentous bacteriophage were spun into continuous microfibers. These fibers can be made out of pure phage solution or a blended solution of

Effect of chloroquine on gene expression of Plasmodium yoelii nigeriensis during its sporogonic development in the mosquito vector

PubMed Central

Silveira, Henrique; Ramos, Susana; Abrantes, Patrícia; Lopes, Luís Filipe; do Rosario, Virgílio E; Abrahamsen, Mitchell S

2007-01-01

Background The anti-malarial chloroquine can modulate the outcome of infection during the Plasmodium sporogonic development, interfering with Plasmodium gene expression and subsequently, with transmission. The present study sets to identify Plasmodium genes that might be regulated by chloroquine in the mosquito vector. Methods Differential display RT-PCR (DDRT-PCR) was used to identify genes expressed during the sporogonic cycle that are regulated by exposure to chloroquine. Anopheles stephensi mosquitoes were fed on Plasmodium yoelii nigeriensis-infected mice. Three days post-infection, mosquitoes were fed a non-infectious blood meal from mice treated orally with 50 mg/kg chloroquine. Two differentially expressed Plasmodium transcripts (Pyn_chl091 and Pyn_chl055) were further characterized by DNA sequencing and real-time PCR analysis. Results Both transcripts were represented in Plasmodium EST databases, but displayed no homology with any known genes. Pyn_chl091 was upregulated by day 18 post infection when the mosquito had a second blood meal. However, when the effect of chloroquine on that transcript was investigated during the erythrocytic cycle, no significant differences were observed. Although slightly upregulated by chloroquine exposure the expression of Pyn_chl055 was more affected by development, increasing towards the end of the sporogonic cycle. Transcript abundance of Pyn_chl055 was reduced when erythrocytic stages were treated with chloroquine. Conclusion Chloroquine increased parasite load in mosquito salivary glands and interferes with the expression of at least two Plasmodium genes. The transcripts identified contain putative signal peptides and transmembrane domains suggesting that these proteins, due to their location, are targets of chloroquine (not as an antimalarial) probably through cell trafficking and recycling. PMID:17605769

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 ...

Development of a workflow for screening and identification of α-amylase inhibitory peptides from food source using an integrated Bioinformatics-phage display approach: Case study - Cumin seed.

PubMed

Siow, Hwee-Leng; Lim, Theam Soon; Gan, Chee-Yuen

2017-01-01

The main objective of this study was to develop an efficient workflow to discover α-amylase inhibitory peptides from cumin seed. A total of 56 unknown peptides was initially found in the cumin seed protein hydrolysate. They were subjected to 2 different in silico screenings and 6 peptides were shortlisted. The peptides were then subjected to in vitro selection using phage display technique and 3 clones (CSP3, CSP4 and CSP6) showed high affinity in binding α-amylase. These clones were subjected to the inhibitory test and only CSP4 and CSP6 exhibited high inhibitory activity. Therefore, these peptides were chemically synthesized for validation purposes. CSP4 exhibited inhibition of bacterial and human salivary α-amylases with IC50 values of 0.11 and 0.04μmol, respectively, whereas CSP6 was about 0.10 and 0.15μmol, respectively. Results showed that the strength of each protocol has been successfully combined as deemed fit to enhance the α-amylase inhibitor peptide discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization.

PubMed

Lemloh, Marie-Louise; Altintoprak, Klara; Wege, Christina; Weiss, Ingrid M; Rothenstein, Dirk

2017-01-28

Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO₂ mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

USDA-ARS?s Scientific Manuscript database

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic...

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.

A Peptide Targeting Inflammatory CNS Lesions in the EAE Rat Model of Multiple Sclerosis.

PubMed

Boiziau, Claudine; Nikolski, Macha; Mordelet, Elodie; Aussudre, Justine; Vargas-Sanchez, Karina; Petry, Klaus G

2018-06-01

Multiple sclerosis is characterized by inflammatory lesions dispersed throughout the central nervous system (CNS) leading to severe neurological handicap. Demyelination, axonal damage, and blood brain barrier alterations are hallmarks of this pathology, whose precise processes are not fully understood. In the experimental autoimmune encephalomyelitis (EAE) rat model that mimics many features of human multiple sclerosis, the phage display strategy was applied to select peptide ligands targeting inflammatory sites in CNS. Due to the large diversity of sequences after phage display selection, a bioinformatics procedure called "PepTeam" designed to identify peptides mimicking naturally occurring proteins was used, with the goal to predict peptides that were not background noise. We identified a circular peptide CLSTASNSC called "Ph48" as an efficient binder of inflammatory regions of EAE CNS sections including small inflammatory lesions of both white and gray matter. Tested on human brain endothelial cells hCMEC/D3, Ph48 was able to bind efficiently when these cells were activated with IL1β to mimic inflammatory conditions. The peptide is therefore a candidate for further analyses of the molecular alterations in inflammatory lesions.

The myeloid-binding peptide adenoviral vector enables multi-organ vascular endothelial gene targeting.

PubMed

Lu, Zhi Hong; Kaliberov, Sergey; Zhang, Jingzhu; Muz, Barbara; Azab, Abdel K; Sohn, Rebecca E; Kaliberova, Lyudmila; Du, Yingqiu; Curiel, David T; Arbeit, Jeffrey M

2014-08-01

Vascular endothelial cells (ECs) are ideal gene therapy targets as they provide widespread tissue access and are the first contact surfaces following intravenous vector administration. Human recombinant adenovirus serotype 5 (Ad5) is the most frequently used gene transfer system because of its appreciable transgene payload capacity and lack of somatic mutation risk. However, standard Ad5 vectors predominantly transduce liver but not the vasculature following intravenous administration. We recently developed an Ad5 vector with a myeloid cell-binding peptide (MBP) incorporated into the knob-deleted, T4 fibritin chimeric fiber (Ad.MBP). This vector was shown to transduce pulmonary ECs presumably via a vector handoff mechanism. Here we tested the body-wide tropism of the Ad.MBP vector, its myeloid cell necessity, and vector-EC expression dose response. Using comprehensive multi-organ co-immunofluorescence analysis, we discovered that Ad.MBP produced widespread EC transduction in the lung, heart, kidney, skeletal muscle, pancreas, small bowel, and brain. Surprisingly, Ad.MBP retained hepatocyte tropism albeit at a reduced frequency compared with the standard Ad5. While binding specifically to myeloid cells ex vivo, multi-organ Ad.MBP expression was not dependent on circulating monocytes or macrophages. Ad.MBP dose de-escalation maintained full lung-targeting capacity but drastically reduced transgene expression in other organs. Swapping the EC-specific ROBO4 for the CMV promoter/enhancer abrogated hepatocyte expression but also reduced gene expression in other organs. Collectively, our multilevel targeting strategy could enable therapeutic biological production in previously inaccessible organs that pertain to the most debilitating or lethal human diseases.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW.

PubMed

Henning, Lisa Maria; Bhatia, Sumati; Bertazzon, Miriam; Marczynke, Michaela; Seitz, Oliver; Volkmer, Rudolf; Haag, Rainer; Freund, Christian

2015-01-01

The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein-protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 μM and 150 µM to the individual WW domains and with a K D of 150 μM to the tandem-WW1-WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome.

Exploring monovalent and multivalent peptides for the inhibition of FBP21-tWW

PubMed Central

Bertazzon, Miriam; Marczynke, Michaela; Seitz, Oliver; Volkmer, Rudolf; Haag, Rainer

2015-01-01

Summary The coupling of peptides to polyglycerol carriers represents an important route towards the multivalent display of protein ligands. In particular, the inhibition of low affinity intracellular protein–protein interactions can be addressed by this design. We have applied this strategy to develop binding partners for FBP21, a protein which is important for the splicing of pre-mRNA in the nucleus of eukaryotic cells. Firstly, by using phage display the optimized sequence WPPPPRVPR was derived which binds with K Ds of 80 μM and 150 µM to the individual WW domains and with a K D of 150 μM to the tandem-WW1–WW2 construct. Secondly, this sequence was coupled to a hyperbranched polyglycerol (hPG) that allowed for the multivalent display on the surface of the dendritic polymer. This novel multifunctional hPG-peptide conjugate displayed a K D of 17.6 µM which demonstrates that the new carrier provides a venue for the future inhibition of proline-rich sequence recognition by FBP21 during assembly of the spliceosome. PMID:26124874

Synthetic vaccines.

PubMed

Lerner, R A

1983-02-01

Synthetic vaccines are designed with the help of computer-graphics programs. These displays generated by Arthur J. Olson of the Research Institute of Scripps Clinic show a method whereby parts of a viral protein that are on the surface of a virus, and therefore accessible to antibodies, can be identified. The backbone of the surface domain of the protein on the outer shell of the tomato bushy-stunt virus is displayed (1) on the basis of coordinates determined by Stephen C. Harrison of Harvard University and his colleagues. A single peptide of the protein is picked out in yellow, with the side chains of its component amino acids indicated in atomic detail (2). The peptide is enlarged and a sphere representing a water molecule is displayed (3). The sphere is rolled around the peptide to generate a map of the surface accessible to water (4); it does so, following an algorithm developed by Michael L. Connolly, by placing a dot at each point of its closest contact with the peptide, taking account of the sphere's own van der Waals radius (zone of influence, in effect) and that of each atom of the peptide and the rest of the protein. A similar-dot-surface map is generated to show what parts of the peptide are still accessible to water when three copies of the protein are associated in an array on the surface of the virus (5) and when four such arrays (out of 60) are in position on the outer surface of the virus (6).

Oxidation of Peptides by Methyl(trifluoromethyl)dioxirane: the Protecting Group Matters

PubMed Central

Rella, Maria Rosaria; Williard, Paul G.

2011-01-01

Representative Boc protected and acetyl protected peptide methyl esters bearing alkyl side chains undergo effective oxidation using methyl(trifluoromethyl)dioxirane (1b) under mild conditions. We observe a protecting group dependency in the chemoselectivity displayed by the dioxirane 1b. N-hydroxylation occurs in the case of the Boc protected peptides, side chain hydroxylation takes place in the case of acetyl protected peptides. Both are attractive transformations since they yield derivatized peptides that serve as valuable synthons. PMID:17221970

Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades.

PubMed

Bidwell, Gene L; Raucher, Drazen

2009-10-01

Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that inhibit signal transduction cascades are discussed. The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. Given our current knowledge of protein sequences, structures and interaction interfaces, therapeutic peptides that inhibit interactions of interest are easily designed. These peptides are advantageous because they are highly specific for the interaction of interest, and they are much more easily developed than small molecule inhibitors of the same interactions. The main hurdle to application of peptides for cancer therapy is their poor pharmacokinetic and biodistribution parameters. Therefore, successful development of peptide delivery vectors could potentially make possible the use of this new and very promising class of anticancer agents.

28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

PubMed

Masias, Emilse; Sanches, Paulo R S; Dupuy, Fernando G; Acuna, Leonardo; Bellomio, Augusto; Cilli, Eduardo; Saavedra, Lucila; Minahk, Carlos

2015-01-01

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

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.

Preinfection of citrus by RB strains of Citrus tristeza virus (CTV) negatively affected expression of a modified T36 CTV vector

USDA-ARS?s Scientific Manuscript database

Genetically modified T36 Citrus tristeza virus (T36-mCTV) is showing promise in Florida to mitigate huanglongbing (HLB) by expressing antimicrobial peptides and RNAi against the presumed pathogen, “Candidatus Liberibacter asiaticus” (CLas), and its vector, the Asian citrus psyllid (ACP). To this end...

Simulated digestion for testing the stability of edible vaccine based on Cucumber mosaic virus (CMV) chimeric particle display Hepatitis C virus (HCV) peptide.

PubMed

Vitti, Antonella; Nuzzaci, Maria; Condelli, Valentina; Piazzolla, Pasquale

2014-01-01

Edible vaccines must survive digestive process and preserve the specific structure of the antigenic peptide to elicit effective immune response. The stability of a protein to digestive process can be predicted by subjecting it to the in vitro assay with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Here, we describe the protocol of producing and using chimeric Cucumber mosaic virus (CMV) displaying Hepatitis C virus (HCV) derived peptide (R9) in double copy as an oral vaccine. Its stability after treatment with SGF and SIF and the preservation of the antigenic properties were verified by SDS-PAGE and immuno western blot techniques.

Radiolabeled Peptide Scaffolds for PET/SPECT - Optical in Vivo Imaging of Carbohydrate-Lectin Interactions

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

Deutscher, Susan

2014-09-30

The objective of this research is to develop phage display-selected peptides into radio- and fluoresecently- labeled scaffolds for the multimodal imaging of carbohydrate-lectin interactions. While numerous protein and receptor systems are being explored for the development of targeted imaging agents, the targeting and analysis of carbohydrate-lectin complexes in vivo remains relatively unexplored. Antibodies, nanoparticles, and peptides are being developed that target carbohydrate-lectin complexes in living systems. However, antibodies and nanoparticles often suffer from slow clearance and toxicity problems. Peptides are attractive alternative vehicles for the specific delivery of radionuclides or fluorophores to sites of interest in vivo, although, because ofmore » their size, uptake and retention may be less than antibodies. We have selected high affinity peptides that bind a specific carbohydrate-lectin complex involved in cell-cell adhesion and cross-linking using bacteriophage (phage) display technologies (1,2). These peptides have allowed us to probe the role of these antigens in cell adhesion. Fluorescent versions of the peptides have been developed for optical imaging and radiolabeled versions have been used in single photon emission computed tomography (SPECT) and positron emission tomography (PET) in vivo imaging (3-6). A benefit in employing the radiolabeled peptides in SPECT and PET is that these imaging modalities are widely used in living systems and offer deep tissue sensitivity. Radiolabeled peptides, however, often exhibit poor stability and high kidney uptake in vivo. Conversely, optical imaging is sensitive and offers good spatial resolution, but is not useful for deep tissue penetration and is semi-quantitative. Thus, multimodality imaging that relies on the strengths of both radio- and optical- imaging is a current focus for development of new in vivo imaging agents. We propose a novel means to improve the efficacy of radiolabeled and fluorescently labeled peptides, including our lectin/carbohydrate- targeting peptides, by displaying the targeting epitopes on small ~29 amino acid cyclic plant protein scaffolds known as cyclotides. Cyclotides are extremely stable molecules with long serum half-lives and low kidney uptake (7). More than one copy of the peptide can be engineered into the cyclotide loops, thus increasing the avidity of the peptide construct for its target.« less

Development of novel recombinant biomimetic chimeric MPG-based peptide as nanocarriers for gene delivery: Imitation of a real cargo.

PubMed

Majidi, Asia; Nikkhah, Maryam; Sadeghian, Faranak; Hosseinkhani, Saman

2016-10-01

In last decades great efforts have been devoted to the study of development of recombinant peptide based vectors that consist of biological motifs with potential applications in gene therapy. Recombinant Biomimetic Chimeric Vectors (rBCVs) are biopolymeric nanocarriers that are designed to mimic viral features to overcome the cellular obstacles in gene transferring pathway into cell nucleus. In this research, we designed and genetically engineered three novel rBCVs with similar sequences that differed in motifs arrangement and motif abundance: MPG-2H1, 2TMPG-2H1 and 2RMPG-2H1. The MPG as a famous amphipathic cell penetrating peptide is the main segment of these constructs which was studied for the first time in association with truncated histone H1 DNA condensing motif. Through the performance of several physicochemical and biological assays, the rBCVs were remarkably examined regarding transfection efficiency. The main objective of this study is focused on the importance of motif design in transfection efficiency of rBCVs on one hand, and the assessment of correlation between structural features and functionality of motifs on the other hand. The results revealed that all three kinds of rBCVs/pDNA nanoparticles with average sizes of 200nm could overwhelm the cellular obstacles associated with gene transfer, and lead to efficient gene delivery. Furthermore, no significant toxicity was perceived and efficient endosome disruptive activity was obtained. It is noteworthy to say among three mentioned constructs 2RMPG-2H1 showed the highest transfection efficiency. Overall the peptide based vectors hold great promise as a nontoxic and effective gene carrier in vitro and in vivo, besides the rational design possibility as the most vital advantages over the other non-viral gene delivery vectors. Copyright © 2016 Elsevier B.V. All rights reserved.

Three methods of presenting flight vector information in a head-up display during simulated STOL approaches

NASA Technical Reports Server (NTRS)

Dwyer, J. H., III; Palmer, E. A., III

1975-01-01

A simulator study was conducted to determine the usefulness of adding flight path vector symbology to a head-up display designed to improve glide-slope tracking performance during steep 7.5 deg visual approaches in STOL aircraft. All displays included a fixed attitude symbol, a pitch- and roll-stabilized horizon bar, and a glide-slope reference bar parallel to and 7.5 deg below the horizon bar. The displays differed with respect to the flight-path marker (FPM) symbol: display 1 had no FPM symbol; display 2 had an air-referenced FPM, and display 3 had a ground-referenced FPM. No differences between displays 1 and 2 were found on any of the performance measures. Display 3 was found to decrease height error in the early part of the approach and to reduce descent rate variation over the entire approach. Two measures of workload did not indicate any differences between the displays.

Gene Transduction and Cell Entry Pathway of Fiber-Modified Adenovirus Type 5 Vectors Carrying Novel Endocytic Peptide Ligands Selected on Human Tracheal Glandular Cells

PubMed Central

Gaden, Florence; Franqueville, Laure; Magnusson, Maria K.; Hong, Saw See; Merten, Marc D.; Lindholm, Leif; Boulanger, Pierre

2004-01-01

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain. PMID:15194799

NHLBI-AbDesigner: an online tool for design of peptide-directed antibodies.

PubMed

Pisitkun, Trairak; Hoffert, Jason D; Saeed, Fahad; Knepper, Mark A

2012-01-01

Investigation of physiological mechanisms at a cellular level often requires production of high-quality antibodies, frequently using synthetic peptides as immunogens. Here we describe a new, web-based software tool called NHLBI-AbDesigner that allows the user to visualize the information needed to choose optimal peptide sequences for peptide-directed antibody production (http://helixweb.nih.gov/AbDesigner/). The choice of an immunizing peptide is generally based on a need to optimize immunogenicity, antibody specificity, multispecies conservation, and robustness in the face of posttranslational modifications (PTMs). AbDesigner displays information relevant to these criteria as follows: 1) "Immunogenicity Score," based on hydropathy and secondary structure prediction; 2) "Uniqueness Score," a predictor of specificity of an antibody against all proteins expressed in the same species; 3) "Conservation Score," a predictor of ability of the antibody to recognize orthologs in other animal species; and 4) "Protein Features" that show structural domains, variable regions, and annotated PTMs that may affect antibody performance. AbDesigner displays the information online in an interactive graphical user interface, which allows the user to recognize the trade-offs that exist for alternative synthetic peptide choices and to choose the one that is best for a proposed application. Several examples of the use of AbDesigner for the display of such trade-offs are presented, including production of a new antibody to Slc9a3. We also used the program in large-scale mode to create a database listing the 15-amino acid peptides with the highest Immunogenicity Scores for all known proteins in five animal species, one plant species (Arabidopsis thaliana), and Saccharomyces cerevisiae.

HIV-1 vaccine strategies utilizing viral vectors including antigen- displayed inoviral vectors.

PubMed

Hassapis, Kyriakos A; Kostrikis, Leondios G

2013-12-01

Antigen-presenting viral vectors have been extensively used as vehicles for the presentation of antigens to the immune system in numerous vaccine strategies. Particularly in HIV vaccine development efforts, two main viral vectors have been used as antigen carriers: (a) live attenuated vectors and (b) virus-like particles (VLPs); the former, although highly effective in animal studies, cannot be clinically tested in humans due to safety concerns and the latter have failed to induce broadly neutralizing anti-HIV antibodies. For more than two decades, Inoviruses (non-lytic bacterial phages) have also been utilized as antigen carriers in several vaccine studies. Inoviral vectors are important antigen-carriers in vaccine development due to their ability to present an antigen on their outer architecture in many copies and to their natural high immunogenicity. Numerous fundamental studies have been conducted, which have established the unique properties of antigen-displayed inoviral vectors in HIV vaccine efforts. The recent isolation of new, potent anti-HIV broadly neutralizing monoclonal antibodies provides a new momentum in this emerging technology.

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.

Constancy and diversity in the flavivirus fusion peptide.

PubMed

Seligman, Stephen J

2008-02-14

Flaviviruses include the mosquito-borne dengue, Japanese encephalitis, yellow fever and West Nile and the tick-borne encephalitis viruses. They are responsible for considerable world-wide morbidity and mortality. Viral entry is mediated by a conserved fusion peptide containing 16 amino acids located in domain II of the envelope protein E. Highly orchestrated conformational changes initiated by exposure to acidic pH accompany the fusion process and are important factors limiting amino acid changes in the fusion peptide that still permit fusion with host cell membranes in both arthropod and vertebrate hosts. The cell-fusing related agents, growing only in mosquitoes or insect cell lines, possess a different homologous peptide. Analysis of 46 named flaviviruses deposited in the Entrez Nucleotides database extended the constancy in the canonical fusion peptide sequences of mosquito-borne, tick-borne and viruses with no known vector to include more recently-sequenced viruses. The mosquito-borne signature amino acid, G104, was also found in flaviviruses with no known vector and with the cell-fusion related viruses. Despite the constancy in the canonical sequences in pathogenic flaviviruses, mutations were surprisingly frequent with a 27% prevalence of nonsynonymous mutations in yellow fever virus fusion peptide sequences, and 0 to 7.4% prevalence in the others. Six of seven yellow fever patients whose virus had fusion peptide mutations died. In the cell-fusing related agents, not enough sequences have been deposited to estimate reliably the prevalence of fusion peptide mutations. However, the canonical sequences homologous to the fusion peptide and the pattern of disulfide linkages in protein E differed significantly from the other flaviviruses. The constancy of the canonical fusion peptide sequences in the arthropod-borne flaviviruses contrasts with the high prevalence of mutations in most individual viruses. The discrepancy may be the result of a survival advantage accompanying sequence diversity (quasispecies) involving the fusion peptide. Limited clinical data with yellow fever virus suggest that the presence of fusion peptide mutants is not associated with a decreased case fatality rate. The cell-fusing related agents may have substantial differences from other flaviviruses in their mechanism of viral entry into the host cell.

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

PubMed Central

Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

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.

Biomimetic graphene sensors: functionalizing graphene with peptides

NASA Astrophysics Data System (ADS)

Ishigami, Masa; Nyon Kim, Sang; Naik, Rajesh; Tatulian, Suren A.; Katoch, Jyoti

2012-02-01

Non-covalent biomimetic functionalization of graphene using peptides is one of more promising methods for producing novel sensors with high sensitivity and selectivity. Here we combine atomic force microscopy, Raman spectroscopy, and attenuated total reflection Fourier transform infrared spectroscopy to investigate peptide binding to graphene and graphite. We choose to study a dodecamer peptide identified with phage display to possess affinities for graphite and we find that the peptide forms a complex mesh-like structure upon adsorption on graphene. Moreover, optical spectroscopy reveals that the peptide binds non-covalently to graphene and possesses an optical signature of an ?-helical conformation on graphene.

Quantitative PET Imaging with Novel HER3 Targeted Peptides Selected by Phage Display to Predict Androgen Independent Prostate Cancer Progression

DTIC Science & Technology

2017-08-01

9 4 1. Introduction The subject of this research is the design and testing of a PET imaging agent for the detection and...AWARD NUMBER: W81XWH-16-1-0447 TITLE: Quantitative PET Imaging with Novel HER3-Targeted Peptides Selected by Phage Display to Predict Androgen...MA 02114 REPORT DATE: August 2017 TYPE OF REPORT: Annual PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland

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.

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.

Evidence of extensive diversity in bacterial adherence mechanisms that exploit unanticipated stainless steel surface structural complexity for biofilm formation.

PubMed

Davis, Elisabeth M; Li, Dongyang; Shahrooei, Mohammad; Yu, Bin; Muruve, Daniel; Irvin, Randall T

2013-04-01

Three protease-resistant bioorganic 304 stainless steel surfaces were created through the reaction of synthetic peptides consisting of the D-enantiomeric isomer (D-K122-4), the retro-inverso D-enantiomeric isomer (RI-K122-4), and a combination of the two peptides (D+RI) of the Pseudomonas aeruginosa PilA receptor binding domain with steel surfaces. The peptides used to produce the new materials differ only in handedness of their three-dimensional structure, but they reacted with the steel to yield materials that differed in their surface electron work function (EWF) while displaying an identical chemical composition and equivalent surface adhesive force properties. These surfaces allowed for an assessment of the relative role of surface EWF in initial biofilm formation. We examined the ability of various bacteria (selected strains of Listeria monocytogenes, L. innocua, Staphylococcus aureus and S. epidermidis) to initiate biofilm formation. The D-K1224 generated surface displayed the lowest EWF (classically associated with greater molecular interactions and more extensive biofilm formation) but was observed to be least effectively colonized by bacteria (>50% decrease in bacterial adherence of all strains). The highest surface EWF with the lowest surface free energy (RI-K122-4 generated) was more extensively colonized by bacteria, with the binding of some strains being equivalent to unmodified steel. The D+RI generated surface was least effective in minimizing biofilm formation, where some strains displayed enhanced bacterial colonization. Fluorescent microscopy revealed that the D and RI peptides displayed similar but clearly different binding patterns, suggesting that the peptides recognized different sites on the steel, and that differential binding of the peptides to the steel surfaces influences the binding of different bacterial strains and species. We have demonstrated that stainless steel surfaces can be easily modified by peptides to generate surfaces with new physiochemical properties. The D-K122-4-modified surface substantially decreases biofilm formation compared to the RI-K122-4 and D+RI surfaces. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Development of a Targeted anti-HER2 scFv Chimeric Peptide for Gene Delivery into HER2-Positive Breast Cancer Cells.

PubMed

Cheraghi, Roya; Nazari, Mahboobeh; Alipour, Mohsen; Majidi, Asia; Hosseinkhani, Saman

2016-12-30

Chimeric polymers are known as suitable carriers for gene delivery. Certain properties are critical for a polymer to be used as a gene delivery vector. A new polymer was designed for the targeted delivery of genes into breast cancer cell lines, based on MPG peptide. It is composed of different functional domains, including HIV gp41, nuclear localization sequence of SV40 T-antigen, two C-terminus repeats of histone H1, and the scFv of anti-HER2 antibody. The results demonstrated that the vector can effectively condense plasmid DNA into nanoparticles with an average size of 250nm. Moreover, fusion of the scFv portion to the carrier brought about the specific recognition of HER2. Overall, the transfection efficiency of the vector demonstrated that it could deliver the desired gene into BT-474 HER2-positive breast cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Nanotube Interactions with Nanoparticles and Peptides

DTIC Science & Technology

2008-01-01

combinatorial phage display technique. We find a tryptophan rich binding motif to nanotubes on solid silicon substrates. The motif resembles an alpha helix...CHAPTER 2. DIELECTROPHORESIS AND PHAGE DISPLAY 2.1. Dielectrophoresis (DEP) 12 2.2. Phage display 14 References...104 5.3. Conclusions 105 5.4. Experimental Section 105 5.4.1. Nanotube synthesis 105 5.4.2. Phage display

Selection of affinity peptides for interference-free detection of cholera toxin.

PubMed

Lim, Jong Min; Heo, Nam Su; Oh, Seo Yeong; Ryu, Myung Yi; Seo, Jeong Hyun; Park, Tae Jung; Huh, Yun Suk; Park, Jong Pil

2018-01-15

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB 5 . Copyright © 2017 Elsevier B.V. All rights reserved.

C. albicans Growth, Transition, Biofilm Formation, and Gene Expression Modulation by Antimicrobial Decapeptide KSL-W

DTIC Science & Technology

2013-11-07

Simon Theberge1, Abdelhabib Semlali1,2, Abdullah Alamri1, Kai P Leung3 and Mahmoud Rouabhia1* Abstract Background: Antimicrobial peptides have been the... peptides , including KSL-W (KKVVFWVKFK-NH2), for potential clinical use. Because this peptide displays antimicrobial activity against bacteria, we sought...the efficacy of KSL-W against C. albicans and its potential use as an antifungal therapy. Keywords: Antimicrobial peptide , KSL-W, C. albicans, Growth

The dominant role of side chains in supramolecular double helical organisation in synthetic tripeptides

NASA Astrophysics Data System (ADS)

Sharma, Ankita; Tiwari, Priyanka; Dutt Konar, Anita

2018-06-01

Peptide self-assembled nanostructures have attracted attention recently owing to their promising applications in diversified avenues. To validate the importance of sidechains in supramolecular architectural stabilization, herein this report describes the self-assembly propensities involving weak interactions in a series of model tripeptides Boc-Xaa-Aib-Yaa-OMe I-IV, (where Xaa = 4-F-Phe/NMeSer/Ile & Yaa = Tyr in peptide I-III respectively and Xaa = 4-F-Phe & Yaa = Ile in peptide IV) differing in terminal side chains. The solid state structural analysis reveals that tripeptide (I) displays supramolecular preference for double helical architecture. However, when slight modification has been introduced in the N-terminal side chains disfavour the double helical organisation (Peptide II and III). Indeed the peptides display sheet like ensemble within the framework. Besides replacement of C-terminal Tyr by Ile in peptide I even do not promote the architecture, emphasizing the dominant role of balance of side chains in stabilizing double helical organisation. The CD measurements, concentration dependant studies, NMR titrations and ROESY spectra are well in agreement with the solid state conformational investigation. Moreover the morphological experiments utilizing FE-SEM, support the heterogeneity present in the peptides. Thus this work may not only hold future promise in understanding the structure and function of neurodegenerative diseases but also assist in rational design of protein modification in biologically active peptides.

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.

Virus activated artificial ECM induces the osteoblastic differentiation of mesenchymal stem cells without osteogenic supplements

PubMed Central

Wang, Jianglin; Wang, Lin; Li, Xin; Mao, Chuanbin

2013-01-01

Biochemical and topographical features of an artificial extracellular matrix (aECM) can direct stem cell fate. However, it is difficult to vary only the biochemical cues without changing nanotopography to study their unique role. We took advantage of two unique features of M13 phage, a non-toxic nanofiber-like virus, to generate a virus-activated aECM with constant ordered ridge/groove nanotopography but displaying different fibronectin-derived peptides (RGD, its synergy site PHSRN, and a combination of RGD and PHSRN). One feature is the self-assembly of phage into a ridge/groove structure, another is the ease of genetically surface-displaying a peptide. We found that the unique ridge/groove nanotopography and the display of RGD and PHSRN could induce the osteoblastic differentiation of mesenchymal stem cells (MSCs) without any osteogenic supplements. The aECM formed through self-assembly and genetic engineering of phage can be used to understand the role of peptide cues in directing stem cell behavior while keeping nanotopography constant. PMID:23393624

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.

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.

RGD peptide-displaying M13 bacteriophage/PLGA nanofibers as cell-adhesive matrices for smooth muscle cells

NASA Astrophysics Data System (ADS)

Shin, Yong Cheol; Lee, Jong Ho; Jin, Oh Seong; Lee, Eun Ji; Jin, Lin Hua; Kim, Chang-Seok; Hong, Suck Won; Han, Dong-Wook; Kim, Chuntae; Oh, Jin-Woo

2015-01-01

Extracellular matrices (ECMs) are network structures that play an essential role in regulating cellular growth and differentiation. In this study, novel nanofibrous matrices were fabricated by electrospinning M13 bacteriophage and poly(lactic- co-glycolic acid) (PLGA) and were shown to be structurally and functionally similar to natural ECMs. A genetically-engineered M13 bacteriophage was constructed to display Arg-Gly-Asp (RGD) peptides on its surface. The physicochemical properties of RGD peptide-displaying M13 bacteriophage (RGD-M13 phage)/PLGA nanofibers were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. We used immunofluorescence staining to confirm that M13 bacteriophages were homogenously distributed in RGD-M13 phage/PLGA matrices. Furthermore, RGD-M13 phage/PLGA nanofibrous matrices, having excellent biocompatibility, can enhance the behaviors of vascular smooth muscle cells. This result suggests that RGD-M13 phage/PLGA nanofibrous matrices have potentials to serve as tissue engineering scaffolds.

Mucosal vaccination by adenoviruses displaying reovirus sigma 1

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

Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. Whenmore » wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.« less

Gender-specific increase of bone mass by CART peptide treatment is ovary-dependent.

PubMed

Gerrits, Han; Bakker, Nicole Ec; van de Ven-de Laat, Cindy Jm; Bourgondien, Freek Gm; Peddemors, Carolien; Litjens, Ralph Hgm; Kok, Han J; Vogel, Gerard Mt; Krajnc-Franken, Magda Am; Gossen, Jan A

2011-12-01

Cocaine- and amphetamine-regulated transcript (CART) has emerged as a neurotransmitter and hormone that has been implicated in many processes including food intake, maintenance of body weight, and reward, but also in the regulation of bone mass. CART-deficient mice are characterized by an osteoporotic phenotype, whereas female transgenic mice overexpressing CART display an increase in bone mass. Here we describe experiments that show that peripheral subcutaneous sustained release of different CART peptide isoforms for a period up to 60 days increased bone mass by 80% in intact mice. CART peptides increased trabecular bone mass, but not cortical bone mass, and the increase was caused by reduced osteoclast activity in combination with normal osteoblast activity. The observed effect on bone was gender-specific, because male mice did not respond to treatment with CART peptides. In addition, male transgenic CART overexpressing mice did not display increased bone mass. Ovariectomy (OVX) completely abolished the increase of bone mass by CART peptides, both in CART peptide-treated wild-type mice and in CART transgenic mice. The effect of CART peptide treatment on trabecular bone was not mediated by 17β-estradiol (E(2)) because supplementation of OVX mice with E(2) could not rescue the effect of CART peptides on bone. Together, these results indicate that sustained release of CART peptides increases bone mass in a gender-specific way via a yet unknown mechanism that requires the presence of the ovary. Copyright © 2011 American Society for Bone and Mineral Research.

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.

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

Visualizing vector field topology in fluid flows

NASA Technical Reports Server (NTRS)

Helman, James L.; Hesselink, Lambertus

1991-01-01

Methods of automating the analysis and display of vector field topology in general and flow topology in particular are discussed. Two-dimensional vector field topology is reviewed as the basis for the examination of topology in three-dimensional separated flows. The use of tangent surfaces and clipping in visualizing vector field topology in fluid flows is addressed.

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.

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.

Viral vectors encoding endomorphins and serine histogranin attenuate neuropathic pain symptoms after spinal cord injury in rats.

PubMed

Nasirinezhad, Farinaz; Gajavelli, Shyam; Priddy, Blake; Jergova, Stanislava; Zadina, James; Sagen, Jacqueline

2015-01-07

The treatment of spinal cord injury (SCI)-induced neuropathic pain presents a challenging healthcare problem. The lack of available robust pharmacological treatments underscores the need for novel therapeutic methods and approaches. Due to the complex character of neuropathic pain following SCI, therapies targeting multiple mechanisms may be a better choice for obtaining sufficient long-term pain relief. Previous studies in our lab showed analgesic effects using combinations of an NMDA antagonist peptide [Ser1]histogranin (SHG), and the mu-opioid peptides endomorphins (EMs), in several pain models. As an alternative to drug therapy, this study evaluated the analgesic potential of these peptides when delivered via gene therapy. Lentiviruses encoding SHG and EM-1 and EM-2 were intraspinally injected, either singly or in combination, into rats with clip compression SCI 2 weeks following injury. Treated animals showed significant reduction in mechanical and thermal hypersensitivity, compared to control groups injected with GFP vector only. The antinociceptive effects of individually injected components were modest, but the combination of EMs and SHG produced robust and sustained antinociception. The onset of the analgesic effects was observed between 1-5 weeks post-injection and sustained without decrement for at least 7 weeks. No adverse effects on locomotor function were observed. The involvement of SHG and EMs in the observed antinociception was confirmed by pharmacologic inhibition using intrathecal injection of either the opioid antagonist naloxone or an anti-SHG antibody. Immunohistochemical analysis showed the presence of SHG and EMs in the spinal cord of treated animals, and immunodot-blot analysis of CSF confirmed the presence of these peptides in injected animals. In a separate group of rats, delayed injection of viral vectors was performed in order to mimic a more likely clinical scenario. Comparable and sustained antinociceptive effects were observed in these animals using the SHG-EMs combination vectors compared to the group with early intervention. Findings from this study support the potential for direct gene therapy to provide a robust and sustained alleviation of chronic neuropathic pain following SCI. The combination strategy utilizing potent mu-opioid peptides with a naturally-derived NMDA antagonist may produce additive or synergistic analgesic effects without the tolerance development for long-term management of persistent pain.

An antimicrobial helix A-derived peptide of heparin cofactor II blocks endotoxin responses in vivo.

PubMed

Papareddy, Praveen; Kalle, Martina; Singh, Shalini; Mörgelin, Matthias; Schmidtchen, Artur; Malmsten, Martin

2014-05-01

Host defense peptides are key components of the innate immune system, providing multi-facetted responses to invading pathogens. Here, we describe that the peptide GKS26 (GKSRIQRLNILNAKFAFNLYRVLKDQ), corresponding to the A domain of heparin cofactor II (HCII), ameliorates experimental septic shock. The peptide displays antimicrobial effects through direct membrane disruption, also at physiological salt concentration and in the presence of plasma and serum. Biophysical investigations of model lipid membranes showed the antimicrobial action of GKS26 to be mirrored by peptide incorporation into, and disordering of, bacterial lipid membranes. GKS26 furthermore binds extensively to bacterial lipopolysaccharide (LPS), as well as its endotoxic lipid A moiety, and displays potent anti-inflammatory effects, both in vitro and in vivo. Thus, for mice challenged with ip injection of LPS, GKS26 suppresses pro-inflammatory cytokines, reduces vascular leakage and infiltration in lung tissue, and normalizes coagulation. Together, these findings suggest that GKS26 may be of interest for further investigations as therapeutic against severe infections and septic shock. Copyright © 2014 Elsevier B.V. All rights reserved.

Peptide-based antibody alternatives for biological sensing in austere environments

NASA Astrophysics Data System (ADS)

Coppock, Matthew B.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

2017-02-01

The most critical component of a biosensor, the biorecognition element, must exhibit high selectivity and strong affinity for a target of interest in operational sensing. Monoclonal antibodies are the current standard reagents for such devices, but their adaptability, manufacturability, and stability greatly limit their effectiveness in fieldable sensors. Peptides have emerged as potential antibody replacements in such applications due to their similar binding performance, extreme chemical and thermal stabilities, and on-demand scalability. In conjunction with modeling capabilities, work at the Army Research Lab focuses on protein catalyzed capture (PCC) agent technology and bacterial display for the discovery of these novel peptide binding reagents. The synthetic, bottom-up PCC agent technology uses an iterative, in situ "click chemistry" approach to produce high performing peptides against specific epitopes translatable to the protein target. Bacterial display allows rapid reagent discovery due to the combination of fast bacterial growth and effective peptide sequence enrichment through multiple rounds of biopanning. Recent advances in both methods are highlighted in regards to the discovery of reagents against Army high priority protein targets for soldier safety, performance, and diagnostics.

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.

Identification of tissue-specific targeting peptide

NASA Astrophysics Data System (ADS)

Jung, Eunkyoung; Lee, Nam Kyung; Kang, Sang-Kee; Choi, Seung-Hoon; Kim, Daejin; Park, Kisoo; Choi, Kihang; Choi, Yun-Jaie; Jung, Dong Hyun

2012-11-01

Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

Structure, Content, and Bioactivity of Food-Derived Peptides in the Body.

PubMed

Sato, Kenji

2018-03-28

Orally administered peptides are assumed to be degraded into amino acids in the body. However, our recent studies revealed some food-derived prolyl and pyroglutamyl peptides with 2-3 amino acid residues in the blood of humans and animals, while most of the peptides in the endoproteinase digest of food protein are degraded by exopeptidase. Some food-derived dipeptides in the body display in vitro and in vivo biological activities. These facts indicate that the biological activities of food-derived peptides in the body rather than those in food are crucial to understanding the mechanism of the beneficial effects of orally administered peptides.

Endoscopic detection of murine colonic dysplasia using a novel fluorescence-labeled peptide

NASA Astrophysics Data System (ADS)

Miller, Sharon J.; Joshi, Bishnu P.; Gaustad, Adam; Fearon, Eric R.; Wang, Thomas D.

2011-03-01

Current endoscopic screening does not detect all pre-malignant (dysplastic) colorectal mucosa, thus requiring the development of more sensitive, targeted techniques to improve detection. The presented work utilizes phage display to identify a novel peptide binder to colorectal dysplasia in a CPC;Apc mouse model. A wide-field, small animal endoscope capable of fluorescence excitation (450-475 nm) identified polyps via white light and also collected fluorescence images (510 nm barrier filter) of peptide binding. The peptide bound ~2-fold greater to the colonic adenomas when compared to the control peptide. We have imaged fluorescence-labeled peptide binding in vivo that is specific towards distal colonic adenomas.

Trypanosoma cruzi TcSMUG L-surface mucins promote development and infectivity in the triatomine vector Rhodnius prolixus.

PubMed

Gonzalez, Marcelo S; Souza, Marcela S; Garcia, Eloi S; Nogueira, Nadir F S; Mello, Cícero B; Cánepa, Gaspar E; Bertotti, Santiago; Durante, Ignacio M; Azambuja, Patrícia; Buscaglia, Carlos A

2013-11-01

TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus.

Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.

PubMed

Suresh, Bharathi; Ramakrishna, Suresh; Kim, Hyongbum

2017-01-01

The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system represents an efficient tool for genome editing. It consists of two components: the Cas9 protein and a guide RNA. To date, delivery of these two components has been achieved using either plasmid or viral vectors or direct delivery of protein and RNA. Plasmid- and virus-free direct delivery of Cas9 protein and guide RNA has several advantages over the conventional plasmid-mediated approach. Direct delivery results in shorter exposure time at the cellular level, which in turn leads to lower toxicity and fewer off-target mutations with reduced host immune responses, whereas plasmid- or viral vector-mediated delivery can result in uncontrolled integration of the vector sequence into the host genome and unwanted immune responses. Cell-penetrating peptide (CPP), a peptide that has an intrinsic ability to translocate across cell membranes, has been adopted as a means of achieving efficient Cas9 protein and guide RNA delivery. We developed a method for treating human cell lines with CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs that leads to endogenous gene disruption. Here we describe a protocol for preparing an efficient CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs, as well as treatment methods to achieve safe genome editing in human cell lines.

Dual genetically encoded phage-displayed ligands.

PubMed

Mohan, Kritika; Weiss, Gregory A

2014-05-15

M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display. Copyright © 2014 Elsevier Inc. All rights reserved.

Enterocin X, a novel two-peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides.

PubMed

Hu, Chih-Bo; Malaphan, Wanna; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-07-01

Enterocin X, composed of two antibacterial peptides (Xalpha and Xbeta), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xalpha and Xbeta display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known.

Importance of lipopolysaccharide aggregate disruption for the anti-endotoxic effects of heparin cofactor II peptides.

PubMed

Singh, Shalini; Papareddy, Praveen; Kalle, Martina; Schmidtchen, Artur; Malmsten, Martin

2013-11-01

Lipid membrane and lipopolysaccharide (LPS) interactions were investigated for a series of amphiphilic and cationic peptides derived from human heparin cofactor II (HCII), using dual polarization interferometry, ellipsometry, circular dichroism (CD), cryoTEM, and z-potential measurements. Antimicrobial effects of these peptides were compared to their ability to disorder bacterial lipid membranes, while their capacity to block endotoxic effects of LPS was correlated to the binding of these peptides to LPS and its lipid A moiety, and to charge, secondary structure, and morphology of peptide/LPS complexes. While the peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) displayed potent antimicrobial and anti-endotoxic effects, its truncated variants KYE21 (KYEITTIHNLFRKLTHRLFRR) and NLF20 (NLFRKLTHRLFRRNFGYTLR) provide some clues on structure-activity relations, since KYE21 retains both the antimicrobial and anti-endotoxic effects of KYE28 (although both attenuated), while NLF20 retains the antimicrobial but only a fraction of the anti-endotoxic effect, hence locating the anti-endotoxic effects of KYE28 to its N-terminus. The antimicrobial effect, on the other hand, is primarily located at the C-terminus of KYE28. While displaying quite different endotoxic effects, these peptides bind to a similar extent to both LPS and lipid A, and also induce comparable LPS scavenging on model eukaryotic membranes. In contrast, fragmentation and densification of LPS aggregates, in turn dependent on the secondary structure in the peptide/LPS aggregates, correlate to the anti-endotoxic effect of these peptides, thus identifying peptide-induced packing transitions in LPS aggregates as key for anti-endotoxic functionality. This aspect therefore needs to be taken into account in the development of novel anti-endotoxic peptide therapeutics. Copyright © 2013. Published by Elsevier B.V.

Isolation, cloning and expression mapping of a gene encoding an anti-diuretic hormone and other CAPA-related peptides in the disease vector, Rhodnius prolixus

USDA-ARS?s Scientific Manuscript database

Following a blood meal, Rhodnius prolixus undergoes a rapid diuresis in order to eliminate excess water and salts. During the voiding of this primary urine, R. prolixus acts as a vector of Chagas’ disease, with the causative agent, Trypanosoma cruzi, infecting the human host via the urine. Diuresi...

Identification of Cyclin A Binders with a Fluorescent Peptide Sensor.

PubMed

Pazos, Elena; Mascareñas, José L; Vázquez, M Eugenio

2016-01-01

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

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.

A direct biocombinatorial strategy toward next generation, mussel-glue inspired saltwater adhesives.

PubMed

Wilke, Patrick; Helfricht, Nicolas; Mark, Andreas; Papastavrou, Georg; Faivre, Damien; Börner, Hans G

2014-09-10

Biological materials exhibit remarkable, purpose-adapted properties that provide a source of inspiration for designing new materials to meet the requirements of future applications. For instance, marine mussels are able to attach to a broad spectrum of hard surfaces under hostile conditions. Controlling wet-adhesion of synthetic macromolecules by analogue processes promises to strongly impact materials sciences by offering advanced coatings, adhesives, and glues. The de novo design of macromolecules to mimic complex aspects of mussel adhesion still constitutes a challenge. Phage display allows material scientists to design specifically interacting molecules with tailored affinity to material surfaces. Here, we report on the integration of enzymatic processing steps into phage display biopanning to expand the biocombinatorial procedure and enable the direct selection of enzymatically activable peptide adhesion domains. Adsorption isotherms and single molecule force spectroscopy show that those de novo peptides mimic complex aspects of bioadhesion, such as enzymatic activation (by tyrosinase), the switchability from weak to strong binders, and adsorption under hostile saltwater conditions. Furthermore, peptide-poly(ethylene oxide) conjugates are synthesized to generate protective coatings, which possess anti-fouling properties and suppress irreversible interactions with blood-plasma protein cocktails. The extended phage display procedure provides a generic way to non-natural peptide adhesion domains, which not only mimic nature but also improve biological sequence sections extractable from mussel-glue proteins. The de novo peptides manage to combine several tasks in a minimal 12-mer sequence and thus pave the way to overcome major challenges of technical wet glues.

Artificial intelligence systems based on texture descriptors for vaccine development.

PubMed

Nanni, Loris; Brahnam, Sheryl; Lumini, Alessandra

2011-02-01

The aim of this work is to analyze and compare several feature extraction methods for peptide classification that are based on the calculation of texture descriptors starting from a matrix representation of the peptide. This texture-based representation of the peptide is then used to train a support vector machine classifier. In our experiments, the best results are obtained using local binary patterns variants and the discrete cosine transform with selected coefficients. These results are better than those previously reported that employed texture descriptors for peptide representation. In addition, we perform experiments that combine standard approaches based on amino acid sequence. The experimental section reports several tests performed on a vaccine dataset for the prediction of peptides that bind human leukocyte antigens and on a human immunodeficiency virus (HIV-1). Experimental results confirm the usefulness of our novel descriptors. The matlab implementation of our approaches is available at http://bias.csr.unibo.it/nanni/TexturePeptide.zip.

Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1

PubMed Central

Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

2015-01-01

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484] PMID:26129676

Infective and inactivated filamentous phage as carriers for immunogenic peptides.

PubMed

Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

2012-07-01

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

Rational and efficient preparation of a chimeric protein containing a tandem dimer of thrombopoietin mimetic peptide fused to human growth hormone in Escherichia coli.

PubMed

Wang, Song; Shen, Mingqiang; Xu, Yang; Chen, Fang; Chen, Mo; Chen, Shilei; Wang, Aiping; Zhang, Zhou; Ran, Xinze; Cheng, Tianmin; Su, Yongping; Wang, Junping

2013-04-01

The 14-mer thrombopoietin mimetic peptide (TMP), especially in the form of dimer, displayed potent megakaryocytopoiesis activity in vitro. However, it is difficult to prepare such short peptide with high bioactivity through gene-engineering approaches. In this study, a chimeric protein containing a tandem dimer of TMP (dTMP) fused to human growth hormone (hGH), a kind of hematopoietic growth factor that activates the same signal pathways as thrombopoietin, was produced in Escherichia coli by soluble expression. By rational utilization of the XmnI and EcoRV restriction sites, a PCR fragment encoding dTMP-GH was inserted into the plasmid vector pMAL-p2X at the position right after Xa factor cleavage site, in frame with maltose-binding protein (MBP) gene. Under optimized conditions, a high-level expression of soluble MBP-dTMP-GH fusion protein was obtained. By application of amylose resin chromatography, Xa factor digestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusion protein was separated. Finally, a relatively high yield of dTMP-GH fusion protein with high purity (>98%) and without redundant amino acid was achieved, as identified by high-performance liquid chromatography, mass spectrometry, and amino acid sequencing. The functional assays showed that dTMP-GH could promote the proliferation of megakaryoblast cells and maturation of murine megakaryocytes derived from bone marrow, in a dose-dependent manner. Moreover, an enhanced effect of dTMP-GH on megakaryocytopoiesis was found as compared with equimolar concentration of dTMP and rhGH. This work provides a new avenue to generate thrombopoietic agents based on TMP.

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.

Gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides

NASA Astrophysics Data System (ADS)

Tao, Yu; Zhang, Yan; Ju, Enguo; Ren, Hui; Ren, Jinsong

2015-07-01

We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments.We here report a facile one-pot synthesis of fluorescent gold nanoclusters (AuNCs) via the peptide biomineralization method, which can elicit specific immunological responses. The as-prepared peptide-protected AuNCs (peptide-AuNCs) display strong red fluorescence, and more importantly, as compared to the peptide alone, the immune stimulatory ability of the resulting peptide-AuNCs can not only be retained, but can also be efficaciously enhanced. Moreover, through a dual-delivery of antigen peptides and cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), the as-prepared peptide-AuNC-CpG conjugates can also act as smart self-vaccines to assist in the generation of high immunostimulatory activity, and be applied as a probe for intracellular imaging. Both in vitro and in vivo studies provide strong evidence that the AuNC-based vaccines may be utilized as safe and efficient immunostimulatory agents that are able to prevent and/or treat a variety of ailments. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr02240a

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

PubMed

Loi, Monica; Di Paolo, Daniela; Soster, Marco; Brignole, Chiara; Bartolini, Alice; Emionite, Laura; Sun, Jessica; Becherini, Pamela; Curnis, Flavio; Petretto, Andrea; Sani, Monica; Gori, Alessandro; Milanese, Marco; Gambini, Claudio; Longhi, Renato; Cilli, Michele; Allen, Theresa M; Bussolino, Federico; Arap, Wadih; Pasqualini, Renata; Corti, Angelo; Ponzoni, Mirco; Marchiò, Serena; Pastorino, Fabio

2013-09-10

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB. By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

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.

Directed Evolution of a Cyclized Peptoid-Peptide Chimera against a Cell-Free Expressed Protein and Proteomic Profiling of the Interacting Proteins to Create a Protein-Protein Interaction Inhibitor.

PubMed

Kawakami, Takashi; Ogawa, Koji; Hatta, Tomohisa; Goshima, Naoki; Natsume, Tohru

2016-06-17

N-alkyl amino acids are useful building blocks for the in vitro display evolution of ribosomally synthesized peptides because they can increase the proteolytic stability and cell permeability of these peptides. However, the translation initiation substrate specificity of nonproteinogenic N-alkyl amino acids has not been investigated. In this study, we screened various N-alkyl amino acids and nonamino carboxylic acids for translation initiation with an Escherichia coli reconstituted cell-free translation system (PURE system) and identified those that efficiently initiated translation. Using seven of these efficiently initiating acids, we next performed in vitro display evolution of cyclized peptidomimetics against an arbitrarily chosen model human protein (β-catenin) cell-free expressed from its cloned cDNA (HUPEX) and identified a novel β-catenin-binding cyclized peptoid-peptide chimera. Furthermore, by a proteomic approach using direct nanoflow liquid chromatography-tandem mass spectrometry (DNLC-MS/MS), we successfully identified which protein-β-catenin interaction is inhibited by the chimera. The combination of in vitro display evolution of cyclized N-alkyl peptidomimetics and in vitro expression of human proteins would be a powerful approach for the high-speed discovery of diverse human protein-targeted cyclized N-alkyl peptidomimetics.

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.

First Attempt To Validate Human IgG Antibody Response to Nterm-34kDa Salivary Peptide as Biomarker for Evaluating Exposure to Aedes aegypti Bites

PubMed Central

Elanga Ndille, Emmanuel; Doucoure, Souleymane; Damien, Georgia; Mouchet, François; Drame, Papa Makhtar; Cornelie, Sylvie; Noukpo, Herbert; Yamadjako, Sandra; Djenontin, Armel; Moiroux, Nicolas; Misse, Dorothee; Akogbeto, Martin; Corbel, Vincent; Henry, Marie-Claire; Chandre, Fabrice; Baldet, Thierry; Remoue, Franck

2012-01-01

Background Much effort is being devoted for developing new indicators to evaluate the human exposure to Aedes mosquito bites and the risk of arbovirus transmission. Human antibody (Ab) responses to mosquito salivary components could represent a promising tool for evaluating the human-vector contact. Methodology/Principal findings To develop a specific biomarker of human exposure to Aedes aegypti bites, we measured IgG Ab response to Ae. aegypti Nterm-34 kDa salivary peptide in exposed children in 7 villages of Southern Benin (West Africa). Results showed that specific IgG response presented high inter-individual heterogeneity between villages. IgG response was associated with rainfall and IgG level increased from dry (low exposure) to rainy (high exposure) seasons. These findings indicate that IgG Ab to Nterm-34 kDa salivary peptide may represent a reliable biomarker to detect variation in human exposure to Ae. aegypti bites. Conclusion/Significance This preliminary study highlights the potential use of Ab response to this salivary peptide for evaluating human exposure to Ae. aegypti. This biomarker could represent a new promising tool for assessing the risk of arbovirus transmission and for evaluating the efficacy of vector control interventions. PMID:23166852

New milk protein-derived peptides with potential antimicrobial activity: an approach based on bioinformatic studies.

PubMed

Dziuba, Bartłomiej; Dziuba, Marta

2014-08-20

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins.

New Milk Protein-Derived Peptides with Potential Antimicrobial Activity: An Approach Based on Bioinformatic Studies

PubMed Central

Dziuba, Bartłomiej; Dziuba, Marta

2014-01-01

New peptides with potential antimicrobial activity, encrypted in milk protein sequences, were searched for with the use of bioinformatic tools. The major milk proteins were hydrolyzed in silico by 28 enzymes. The obtained peptides were characterized by the following parameters: molecular weight, isoelectric point, composition and number of amino acid residues, net charge at pH 7.0, aliphatic index, instability index, Boman index, and GRAVY index, and compared with those calculated for known 416 antimicrobial peptides including 59 antimicrobial peptides (AMPs) from milk proteins listed in the BIOPEP database. A simple analysis of physico-chemical properties and the values of biological activity indicators were insufficient to select potentially antimicrobial peptides released in silico from milk proteins by proteolytic enzymes. The final selection was made based on the results of multidimensional statistical analysis such as support vector machines (SVM), random forest (RF), artificial neural networks (ANN) and discriminant analysis (DA) available in the Collection of Anti-Microbial Peptides (CAMP database). Eleven new peptides with potential antimicrobial activity were selected from all peptides released during in silico proteolysis of milk proteins. PMID:25141106

POPISK: T-cell reactivity prediction using support vector machines and string kernels

PubMed Central

2011-01-01

Background Accurate prediction of peptide immunogenicity and characterization of relation between peptide sequences and peptide immunogenicity will be greatly helpful for vaccine designs and understanding of the immune system. In contrast to the prediction of antigen processing and presentation pathway, the prediction of subsequent T-cell reactivity is a much harder topic. Previous studies of identifying T-cell receptor (TCR) recognition positions were based on small-scale analyses using only a few peptides and concluded different recognition positions such as positions 4, 6 and 8 of peptides with length 9. Large-scale analyses are necessary to better characterize the effect of peptide sequence variations on T-cell reactivity and design predictors of a peptide's T-cell reactivity (and thus immunogenicity). The identification and characterization of important positions influencing T-cell reactivity will provide insights into the underlying mechanism of immunogenicity. Results This work establishes a large dataset by collecting immunogenicity data from three major immunology databases. In order to consider the effect of MHC restriction, peptides are classified by their associated MHC alleles. Subsequently, a computational method (named POPISK) using support vector machine with a weighted degree string kernel is proposed to predict T-cell reactivity and identify important recognition positions. POPISK yields a mean 10-fold cross-validation accuracy of 68% in predicting T-cell reactivity of HLA-A2-binding peptides. POPISK is capable of predicting immunogenicity with scores that can also correctly predict the change in T-cell reactivity related to point mutations in epitopes reported in previous studies using crystal structures. Thorough analyses of the prediction results identify the important positions 4, 6, 8 and 9, and yield insights into the molecular basis for TCR recognition. Finally, we relate this finding to physicochemical properties and structural features of the MHC-peptide-TCR interaction. Conclusions A computational method POPISK is proposed to predict immunogenicity with scores which are useful for predicting immunogenicity changes made by single-residue modifications. The web server of POPISK is freely available at http://iclab.life.nctu.edu.tw/POPISK. PMID:22085524

POPISK: T-cell reactivity prediction using support vector machines and string kernels.

PubMed

Tung, Chun-Wei; Ziehm, Matthias; Kämper, Andreas; Kohlbacher, Oliver; Ho, Shinn-Ying

2011-11-15

Accurate prediction of peptide immunogenicity and characterization of relation between peptide sequences and peptide immunogenicity will be greatly helpful for vaccine designs and understanding of the immune system. In contrast to the prediction of antigen processing and presentation pathway, the prediction of subsequent T-cell reactivity is a much harder topic. Previous studies of identifying T-cell receptor (TCR) recognition positions were based on small-scale analyses using only a few peptides and concluded different recognition positions such as positions 4, 6 and 8 of peptides with length 9. Large-scale analyses are necessary to better characterize the effect of peptide sequence variations on T-cell reactivity and design predictors of a peptide's T-cell reactivity (and thus immunogenicity). The identification and characterization of important positions influencing T-cell reactivity will provide insights into the underlying mechanism of immunogenicity. This work establishes a large dataset by collecting immunogenicity data from three major immunology databases. In order to consider the effect of MHC restriction, peptides are classified by their associated MHC alleles. Subsequently, a computational method (named POPISK) using support vector machine with a weighted degree string kernel is proposed to predict T-cell reactivity and identify important recognition positions. POPISK yields a mean 10-fold cross-validation accuracy of 68% in predicting T-cell reactivity of HLA-A2-binding peptides. POPISK is capable of predicting immunogenicity with scores that can also correctly predict the change in T-cell reactivity related to point mutations in epitopes reported in previous studies using crystal structures. Thorough analyses of the prediction results identify the important positions 4, 6, 8 and 9, and yield insights into the molecular basis for TCR recognition. Finally, we relate this finding to physicochemical properties and structural features of the MHC-peptide-TCR interaction. A computational method POPISK is proposed to predict immunogenicity with scores which are useful for predicting immunogenicity changes made by single-residue modifications. The web server of POPISK is freely available at http://iclab.life.nctu.edu.tw/POPISK.

Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

PubMed Central

Marcozzi, Alessio; Masini, Tiziana; Zhu, Di; Pesce, Diego; Illarionov, Boris; Fischer, Markus

2017-01-01

Abstract Enzymes of the 2‐C‐methyl‐d‐erythritol‐4‐phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1‐deoxy‐d‐xylulose‐5‐phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false‐positive hits. The enriched peptide binder P12 emerged as a substrate (d‐glyceraldehyde‐3‐phosphate)‐competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor‐ and acceptor‐substrate‐binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition. PMID:29119720

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

PubMed Central

Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

2008-01-01

Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

[Construction of Plasmodium falciparum signal peptide peptidase-GFP mutant and its expression analysis in the malaria parasite].

PubMed

Li, Xue-rong; Wu, Yin-juan; Shang, Mei; Li, Ye; Xu, Jin; Yu, Xin-bing; Athar, Chishti

2014-08-01

To construct recombinant plasmid pSPPcGT which contains signal peptide peptidase gene of Plasmodium falciparum (PJSPP) and GFP, and transfect into P. falciparum (3D7 strain) to obtain mutant parasites which can express PJSPP-GFP. Plasmodium falciparum(3D7 strain) genomic DNA was extracted from cultured malaria parasites. The C-terminal region of PJSPP, an 883 bp gene fragment was amplified by PCR, and then cloned into pPM2GT vector to get recombinant vector pSPPcGT. The recombinant vectors were identified by PCR, double restriction enzyme digestion and DNA sequencing. pSPPcGT vector was transfected into malaria parasites. The positive clones were selected by adding inhibitor of Plasmodium falciparum dihydrofolate reductase WR99210 to the culture medium. The pSPP-GFP-transfected parasites were fixed with methanol, stained with DAPI, and observed under immunofluorescence microscope. The PJSPP-GFP expression in P. falciparum was identified by SDS-PAGE and Western blotting. The C-terminal region of PJSPP was amplified from P.falciparum (3D7 strain) genomic DNA by PCR with the length of 883 bp. The constructed recombinant vectors were identified by PCR screening, double restriction enzyme digestion and DNA sequencing. The pSPPcGT vector was transfected into P. falciparum and the positive clones were selected by WR99210. GFP fluorescence was observed in transfected parasites by immunofluorescence microscopy, and mainly located in the cytoplasm. The PJSPP-GFP expression in malaria parasites was confirmed by Western blotting with a relative molecular mass of Mr 64,000. Recombinant vector PJSPP-GFP is constructed and transfected into P. falciparum to obtain P. falciparum mutant clone which can express PfSPP-GFP.

Immunomagnetic separation of human myeloperoxidase using an antibody-mimicking peptide identified by phage display.

PubMed

Yun, Soi; Ryu, Hyunmin; Lee, E K

2017-09-10

Phage display biopanning is a powerful in vitro selection process for screening and identifying peptides that bind to a target protein of interest. With the aim of replacing antibodies in immuno-diagnostic applications, we identified peptides whose binding characteristics mimicked those of anti-human myeloperoxidase (hMPO), a biomarker for acute cardiac diseases. Based on ELISA results from four phage clones, we selected and chemically synthesized a 12-mer peptide (SYIEPPERHRHR). Quartz crystal microbalance and surface plasmon resonance analyses revealed that the molar binding equilibrium ratio of the synthesized peptide was 0.023, approximately 43-fold lower than that of the anti-hMPO antibody. The dissociation constant (K d ) was 57nM, which was comparable to that of the native antibody (83nM). Next, we biotinylated the peptide at its N-terminus and attached the biotinylated peptide to the surface of streptavidin-coated magnetic particles to assess its ability to selectively capture hMPO. The binding equilibrium data were similar to the previous analyses; specifically, around 0.021mol peptide bound to 1mol of hMPO. Antigen capture was found to be selective and to be relatively little influenced by the presence of human serum albumin (HSA), an abundant constituent of serum. Our work demonstrates the potential of immunomagnetic isolation to achieve selective capture of a low-concentration antigen from complex solutions such as serum. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Phosphorylation-dependent mineral-type specificity for apatite-binding peptide sequences.

PubMed

Addison, William N; Miller, Sharon J; Ramaswamy, Janani; Mansouri, Ahmad; Kohn, David H; McKee, Marc D

2010-12-01

Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation--a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of the VTKHLNQISQSY peptide (VTK peptide) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled phosphorylated VTK peptide (pVTK) did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral-binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion, phosphorylation of serine residues increases peptide specificity for bone-like mineral, whose adsorption is determined primarily by sequence composition and net charge as opposed to sequence order. However, sequence order in addition to net charge modulates the mineralization of osteoblast cultures. The ability of such peptides to inhibit mineralization has potential utility in the management of pathologic calcification. Copyright © 2010 Elsevier Ltd. 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.

Effect of a cecropin-like synthetic peptide (Shiva-3) on the sporogonic development of Plasmodium berghei.

PubMed

Rodriguez, M C; Zamudio, F; Torres, J A; Gonzalez-Ceron, L; Possani, L D; Rodriguez, M H

1995-06-01

The effect of a synthetic cecropin-like peptide, Shiva-3, on in vitro ookinete development and on the early sporogonic stages of Plasmodium berghei in the midgut of Anopheles albimanus was investigated. Peptide concentrations of 75 and 100 microM were effective (P < 0.05) in reducing ookinete production and the number of infected mosquitoes in almost all experiments. These peptide concentrations in the midgut were not toxic for the survival of the mosquitoes. Complete inhibition was obtained if 100 microM Shiva-3 was applied in the first 8 hr of parasite development. The deleterious effect of the peptide on the parasite was effective after exposure for as short as 50 sec and the permanence of free peptide in the mosquito midgut was estimated to be of a minimum of 5 min. These observations indicate the possibility of using Shiva-like peptide genes to engineering malaria-resistant vectors as an alternative in malaria control strategies.

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.

Directed surface attachment of nanomaterials via coiled-coil-driven self-assembly

NASA Astrophysics Data System (ADS)

White, Simon J.; Johnson, Steven; Szymonik, Michal; Wardingley, Richard A.; Pye, Douglas; Davies, A. Giles; Wälti, Christoph; Stockley, Peter G.

2012-12-01

Numerous nanoscale devices and materials have been fabricated in recent years using a variety of biological scaffolds. However, the interfacing of these devices and materials into existing circuits and ordered arrays has proved problematic. Here, we describe a simple solution to this problem using self-assembly of the peptide coiled-coil heterodimer ACID:BASE to immobilize M13 bacteriophage particles to specific locations on a patterned gold surface. Surface plasmon resonance demonstrated that free ACID peptides will assemble onto a surface derivatized with BASE. We then displayed the ACID peptide on the pIX coat protein of M13 and showed that these phage particles permit formation of the coiled-coil resulting in specific surface attachment. The ACID:immobilized BASE affinities appear to be similar for free peptide and phage-displayed ACID. Finally, we fabricated two gold electrodes, separated by a 200 nm gap, coated one of them with BASE and showed that this allows localization of the M13:ACID onto the functionalized electrode.

Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation

PubMed Central

So, Christopher R.; Hayamizu, Yuhei; Yazici, Hilal; Gresswell, Carolyn; Khatayevich, Dmitriy; Tamerler, Candan; Sarikaya, Mehmet

2012-01-01

Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio-solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino-acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered via simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides via their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio-solid interfaces. PMID:22233341

Establishment of a reliable dual-vector system for the phage display of antibody fragments.

PubMed

Joo, Hyun-yoo; Hur, Byung-ung; Lee, Kyung-woo; Song, Suk-yoon; Cha, Sang-hoon

2008-04-20

To resolve some of the technical limitations in a phage-displayed Fab library, we have designed two dual-vector systems, DVS-I and DVS-II, composed of a set of replicon-compatible plasmid (pLA-1 or pLT-2) for producing soluble L chain fragments and phagemid (pHf1g3T-1 or pHf1g3A-2) for expressing Fd (V(H)+C(H1))-DeltapIII fusion molecules as well as a genotype-phenotype linkage. Compared to the DVS-I (pLA-1 and pHf1g3T-1), the DVS-II (pLT-2 and pHf1g3A-2) showed stable transformation efficiency regardless of the order of the vectors introduced into the host cells. In addition, expression of soluble Fab molecules with antigen-binding reactivity, recombinant phage titer and display level of functional Fab-DeltapIII on the phage progenies of the DVS-II were comparable with a conventional phage display system using a single phagemid vector. More importantly, the phage displaying target-specific Fab-DeltapIII molecules was successfully enriched by panning, which allows isolation of the pHf1g3A-2 phagemid encoding antigen-specific Fd molecules. We believe that the DVS-II may provide a valuable tool in the construction of a combinatorial phage-displayed Fab library with large diversity. Furthermore, it can be readily applied to isolation of desired antibody clones if L chain promiscuity of antibodies in determining antigen-binding specificity is considered, or in guided-selection or chain shuffling of mAbs of non-human origin.

Self-Assembly of an α-Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework

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

Magnotti, Elizabeth L.; Hughes, Spencer A.; Dillard, Rebecca S.

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonalmore » packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. In conclusion, the resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.« less

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.

Self-Assembly of an α-Helical Peptide into a Crystalline Two-Dimensional Nanoporous Framework

DOE PAGES

Magnotti, Elizabeth L.; Hughes, Spencer A.; Dillard, Rebecca S.; ...

2016-11-22

Sequence-specific peptides have been demonstrated to self-assemble into structurally defined nanoscale objects including nanofibers, nanotubes, and nanosheets. The latter structures display significant promise for the construction of hybrid materials for functional devices due to their extended planar geometry. Realization of this objective necessitates the ability to control the structural features of the resultant assemblies through the peptide sequence. The design of a amphiphilic peptide, 3FD-IL, is described that comprises two repeats of a canonical 18 amino acid sequence associated with straight α-helical structures. Peptide 3FD-IL displays 3-fold screw symmetry in a helical conformation and self-assembles into nanosheets based on hexagonalmore » packing of helices. Biophysical evidence from TEM, cryo-TEM, SAXS, AFM, and STEM measurements on the 3FD-IL nanosheets support a structural model based on a honeycomb lattice, in which the length of the peptide determines the thickness of the nanosheet and the packing of helices defines the presence of nanoscale channels that permeate the sheet. The honeycomb structure can be rationalized on the basis of geometrical packing frustration in which the channels occupy defect sites that define a periodic superlattice. In conclusion, the resultant 2D materials may have potential as materials for nanoscale transport and controlled release applications.« less

Peptides design based on transmembrane Escherichia coli's OmpA protein through molecular dynamics simulations in water-dodecane interfaces.

PubMed

Aguilera-Segura, Sonia M; Núñez Vélez, Vanessa; Achenie, Luke; Álvarez Solano, Oscar; Torres, Rodrigo; González Barrios, Andrés Fernando

2016-07-01

Recent research efforts have focused on the production of environmentally nonthreatening products, including identifying biosurfactants that can replace conventional surfactants. In order to utilize biosurfactants in different industries such as cosmetic, food or petroleum, it is necessary to understand the underpinnings behind the interactions that could take place for biosurfactants which display potential for interface activity. This work aimed to use molecular dynamics simulations to understand the interactions of rationally obtained peptide sequences from the original sequence of the OmpA gene in Escherichia coli, based on the free energy change (ΔG) during peptide insertion at the water-dodecane interface. Seventeen OmpA-based peptide sequences were selected and analyzed based on their hydropathy index profiles. We found that free energy change due to Columbic interactions and SASA (ΔGCoul/SASA), total free energy change and MW (ΔG/MW), and free energy change due to Coulombic and van der Waals interactions (ΔGCoul/ΔGvdW) ratios could provide a better understating in the contribution of the free energy decrease at the interface. The results indicated that the peptide sequences GKNHDTGVSPVFA and THENQLGAGAFG display biosurfactant potential based on low ΔG per square nanometer, high ΔGCoul/ΔGvdW ratio, clearly defined moieties along its hydrophobic surface and sequence, and the presence of charged residues in the polar head. Clearly defined moieties and SASA were determinant for electrostatic interactions between oil-water interfaces. Experimental validations exhibited that the emulsions prepared remained stable between 3 and 27h, respectively. Even though the peptide GKNHDTGVSPVFA displays strong interactions at the interface, stabilization times showed that the peptide THENQLGAGAFG exhibited the best performance suggesting that the stability can be better described by kinetic rather than thermodynamic criteria once the emulsion is formed. Copyright © 2016 Elsevier Inc. All rights reserved.

Inorganic binding peptides designed by phage display techniques for biotechnology applications

NASA Astrophysics Data System (ADS)

Liao, Chih-Wei

Biomacromolecules play an important role in the control of hard tissue structure and function via specific molecular recognition interactions between proteins of the matrix and inorganic species of the biomineral phase. During the construction of the tissue, biomacromolecules are usually folded into a certain comformation, analogous to a "lock" for fitting with other proteins or smaller molecules as a "key". Currently, the rational design of molecular recognition in biomacro-molecules is still hard to accomplish because the protein conformation is too complex to precisely predict based on the existing conformational information of proteins found in biological systems. In the past two decades, the combinatorial approach (e.g. phage display techniques) has been used to select short binding peptides with molecular recognition to an inorganic target material without a prior knowledge of the amino acid sequence required for the specific binding. The technique has been referred to as "biopanning" because bacteriophages are used to "screen" for peptides that exhibit strong binding to a target material of interest. In this study, two diverse applications were chosen to demonstrate the utility of the biopanning approach. In one project, phage display techniques were used to pan for Indium Zinc Oxide (InZnO) binding peptides to serve as linkers between transducer devices and biosensing elements for demonstration of the feasibility of reversibly electro-activated biosensors. The amorphous InZnO, with its homogeneous surface, led to three consensus peptide sequences, AGFPNSTHSSNL, SHAPDSTWFALF, and TNSSSQFVVAIP. In addition, it was demonstrated that some selected phage clones of the InZnO binding peptides were able to be released from the InZnO surface after applying a voltage of 1400 mV on an electro-activated releasing device. In the second project, phage display techniques were used to select phage clones that bind specifically to francolite mineral in order to achieve separation of francolite particles from dolomitic particles within Florida phosphate ore. A phage clone with a 12-mer francolite binding peptide of WSITTYHDRAIV was able to concentrate the content of francolite from 25% to 42% in a bench-top flotation process of mixed minerals. The first system demonstrates an advanced technology application of the biopanning approach for the development of novel biosensors, while the second system demonstrates application of the biotechnology approach to a commodity industry.

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

Refactored M13 Bacteriophage as a Platform for Tumor Cell Imaging and Drug Delivery

PubMed Central

MOSER, FELIX; ENDY, DREW; BELCHER, ANGELA M.

2014-01-01

M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as “refactoring,” we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to reengineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications. PMID:23656279

Refactored M13 bacteriophage as a platform for tumor cell imaging and drug delivery.

PubMed

Ghosh, Debadyuti; Kohli, Aditya G; Moser, Felix; Endy, Drew; Belcher, Angela M

2012-12-21

M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as "refactoring," we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to re-engineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications.

Orexin (hypocretin) gene transfer diminishes narcoleptic sleep behavior in mice

PubMed Central

Liu, Meng; Thankachan, Stephen; Kaur, Satvinder; Begum, Suraiya; Blanco-Centurion, Carlos; Sakurai, Takeshi; Yanagisawa, Masashi; Neve, Rachael; Shiromani, Priyattam J.

2008-01-01

Gene transfer has proven to be an effective neurobiological tool in a number of neurodegenerative diseases, but it is not known if it can correct a sleep disorder. Narcolepsy is a neurodegenerative sleep disorder linked to the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Here, a replication-defective herpes simplex virus-1 amplicon-based vector was constructed to transfer the gene for mouse prepro-orexin into mice with a genetic deletion of the orexin gene. After in vitro tests confirmed successful gene transfer into cells, the gene vector was delivered to the lateral hypothalamus of orexin knockout (KO) mice where the orexin peptide was robustly expressed in the somata and processes of numerous neurons, and the peptide product was detected in the cerebrospinal fluid. During the 4-day life-span of the vector the incidence of cataplexy declined by 60%, and the levels of rapid eye movement sleep during the second half of the night were similar to levels in wild-type mice, indicating that narcoleptic sleep–wake behavior in orexin KO mice can be improved by targeted gene transfer. PMID:18973565

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.

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.

Computer Graphics in Research: Some State -of-the-Art Systems

ERIC Educational Resources Information Center

Reddy, R.; And Others

1975-01-01

A description is given of the structure and functional characteristics of three types of interactive computer graphic systems, developed by the Department of Computer Science at Carnegie-Mellon; a high-speed programmable display capable of displaying 50,000 short vectors, flicker free; a shaded-color video display for the display of gray-scale…

Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands.

PubMed

Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2011-06-01

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

Programmable Bio-surfaces for Biomedical Applications.

PubMed

Shiba, Kiyotaka

2017-01-01

A peptide can be used as a functional building block to construct artificial systems when it has sufficient transplantability and functional independence in terms of its assigned function. Recent advances in in vitro evolution systems have been increasing the list of peptides that specifically bind to certain targets, such as proteins and cells. By properly displaying these peptides on solid surfaces, we can endow the inorganic materials with various biological functions, which will contribute to the development of diagnosis and therapeutic medical devices. Here, the methods for the peptide-based surface functionalization are reviewed by focusing on sources of peptides as well as methods of immobilization.

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.

Expanding the versatility of phage display I: efficient display of peptide-tags on protein VII of the filamentous phage.

PubMed

Løset, Geir Åge; Bogen, Bjarne; Sandlie, Inger

2011-02-24

Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS(6) or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations.

Immunogenicity of T7 bacteriophage nanoparticles displaying G-H loop of foot-and-mouth disease virus (FMDV).

PubMed

Xu, Hai; Bao, Xi; Lu, Yu; Liu, Yamei; Deng, Bihua; Wang, Yiwei; Xu, Yue; Hou, Jibo

2017-06-01

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses worldwide. The G-H loop of the FMDV VP1 structural protein is the major neutralizing antigenic site. However, a fully protective G-H loop peptide vaccine requires the addition of promiscuous Th sites from a source outside VP1. Thus, we demonstrated the potential of T7 bacteriophage based nanoparticles displaying a genetically fused G-H loop peptide (T7-GH) as a FMDV vaccine candidate. Recombinant T7-GH phage was constructed by inserting the G-H loop coding region into the T7 Select 415-1b vector. Purified T7-GH phage nanoparticles were analyzed by SDS-PAGE, Western blot and Dot-ELISA. Pigs seronegative for FMDV exposure were immunized with T7-GH nanoparticles along with the adjuvant Montanide ISA206, and two commercially available FMDV vaccines (InactVac and PepVac). Humoral and cellular immune responses, as well as protection against virulent homologous virus challenge were assessed following single dose immunization. Pigs immunized T7-GH developed comparable anti-VP1 antibody titers to PepVac, although lower LPBE titers than was induced by InactVac. Antigen specific lymphocyte proliferation was detected in T7-GH group similar to that of PepVac group, however, weaker than InactVac group. Pigs immunized with T7-GH developed a neutralizing antibody response stronger than PepVac, but weaker than InactVac. Furthermore, 80% (4/5) of T7-GH immunized pigs were protected from challenge with virulent homologous virus. These findings demonstrate that the T7-GH phage nanoparticles were effective in eliciting antigen specific immune responses in pigs, highlighting the value of such an approach in the research and development of FMDV vaccines. 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.

Generation of a novel monoclonal antibody that recognizes the alpha (α)-amidated isoform of a valine residue.

PubMed

Antón Palma, Benito; Leff Gelman, Philippe; Medecigo Ríos, Mayra; Calva Nieves, Juan Carlos; Acevedo Ortuño, Rodolfo; Matus Ortega, Maura Epifanía; Hernández Calderón, Jorge Alberto; Hernández Miramontes, Ricardo; Flores Zamora, Anabel; Salazar Juárez, Alberto

2015-10-13

Alpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species. The α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (-CONH2) and free α-carboxylic acid (-COO(-)) isovariant of the valine residue. P18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/μg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb. Our results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.

Peptide-mediated vectorization of metal complexes: conjugation strategies and biomedical applications.

PubMed

Soler, Marta; Feliu, Lidia; Planas, Marta; Ribas, Xavi; Costas, Miquel

2016-08-16

The rich chemical and structural versatility of transition metal complexes provides numerous novel paths to be pursued in the design of molecules that exert particular chemical or physicochemical effects that could operate over specific biological targets. However, the poor cell permeability of metallodrugs represents an important barrier for their therapeutic use. The conjugation between metal complexes and a functional peptide vector can be regarded as a versatile and potential strategy to improve their bioavailability and accumulation inside cells, and the site selectivity of their effect. This perspective lies in reviewing the recent advances in the design of metallopeptide conjugates for biomedical applications. Additionally, we highlight the studies where this approach has been directed towards the incorporation of redox active metal centers into living organisms for modulating the cellular redox balance, as a tool with application in anticancer therapy.

Trypanosoma cruzi TcSMUG L-surface Mucins Promote Development and Infectivity in the Triatomine Vector Rhodnius prolixus

PubMed Central

Gonzalez, Marcelo S.; Souza, Marcela S.; Garcia, Eloi S.; Nogueira, Nadir F. S.; Mello, Cícero B.; Cánepa, Gaspar E.; Bertotti, Santiago; Durante, Ignacio M.; Azambuja, Patrícia; Buscaglia, Carlos A.

2013-01-01

Background TcSMUG L products were recently identified as novel mucin-type glycoconjugates restricted to the surface of insect-dwelling epimastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. The remarkable conservation of their predicted mature N-terminal region, which is exposed to the extracellular milieu, suggests that TcSMUG L products may be involved in structural and/or functional aspects of the interaction with the insect vector. Methodology and Principal Findings Here, we investigated the putative roles of TcSMUG L mucins in both in vivo development and ex vivo attachment of epimastigotes to the luminal surface of the digestive tract of Rhodnius prolixus. Our results indicate that the exogenous addition of TcSMUG L N-terminal peptide, but not control T. cruzi mucin peptides, to the infected bloodmeal inhibited the development of parasites in R. prolixus in a dose-dependent manner. Pre-incubation of insect midguts with the TcSMUG L peptide impaired the ex vivo attachment of epimastigotes to the luminal surface epithelium, likely by competing out TcSMUG L binding sites on the luminal surface of the posterior midgut, as revealed by fluorescence microscopy. Conclusion and Significance Together, these observations indicate that TcSMUG L mucins are a determinant of both adhesion of T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine, and the infection of the insect vector, R. prolixus. PMID:24244781

Structure-activity relationships in beta-defensin peptides.

PubMed

Taylor, Karen; Barran, Perdita E; Dorin, Julia R

2008-01-01

The beta-defensins comprise a large family of small cationic antimicrobial peptides widely distributed in plants, mammals and insects. These cysteine rich peptides display multifunctional properties with implications as potential therapeutic agents. Recent research has highlighted their role in both the innate and adaptive immune systems as well as being novel melanocortin ligands. Studies investigating structure and function provide an insight into the molecular basis of their immunological properties. (c) 2007 Wiley Periodicals, Inc.

Synthesis of tumor necrosis factor α for use as a mirror-image phage display target.

PubMed

Petersen, Mark E; Jacobsen, Michael T; Kay, Michael S

2016-06-21

Tumor Necrosis Factor alpha (TNFα) is an inflammatory cytokine that plays a central role in the pathogenesis of chronic inflammatory disease. Here we describe the chemical synthesis of l-TNFα along with the mirror-image d-protein for use as a phage display target. The synthetic strategy utilized native chemical ligation and desulfurization to unite three peptide segments, followed by oxidative folding to assemble the 52 kDa homotrimeric protein. This synthesis represents the foundational step for discovering an inhibitory d-peptide with the potential to improve current anti-TNFα therapeutic strategies.

Application of phage peptide display technology for the study of food allergen epitopes.

PubMed

Chen, Xueni; Dreskin, Stephen C

2017-06-01

Phage peptide display technology has been used to identify IgE-binding mimotopes (mimics of natural epitopes) that mimic conformational epitopes. This approach is effective in the characterization of those epitopes that are important for eliciting IgE-mediated allergic responses by food allergens and those that are responsible for cross-reactivity among allergenic food proteins. Application of this technology will increase our understanding of the mechanisms whereby food allergens elicit allergic reactions, will facilitate the discovery of diagnostic reagents and may lead to mimotope-based immunotherapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus.

PubMed

Klausberger, Miriam; Tscheliessnig, Rupert; Neff, Silke; Nachbagauer, Raffael; Wohlbold, Teddy John; Wilde, Monika; Palmberger, Dieter; Krammer, Florian; Jungbauer, Alois; Grabherr, Reingard

2016-01-01

Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection.

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.

Vector breather-to-soliton transitions and nonlinear wave interactions induced by higher-order effects in an erbium-doped fiber

NASA Astrophysics Data System (ADS)

Sun, Wen-Rong; Wang, Lei; Xie, Xi-Yang

2018-06-01

Vector breather-to-soliton transitions for the higher-order nonlinear Schrödinger-Maxwell-Bloch (NLS-MB) system with sextic terms are investigated. The Lax pair and Darboux transformation (DT) of such system are constructed. With the DT, analytic vector breather solutions up to the second order are obtained. With appropriate choices of the spectra parameters, vector breather-to-soliton transitions happen. Interaction mechanisms of vector nonlinear waves (breather-soliton or soliton-soliton interactions) are displayed.

Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-gamma-cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector.

PubMed

Huang, Hongliang; Yu, Hai; Tang, Guping; Wang, Qingqing; Li, Jun

2010-03-01

Gene delivery is one of the critical steps for gene therapy. Non-viral vectors have many advantages but suffered from low gene transfection efficiency. Here, in order to develop new polymeric gene vectors with low cytotoxicity and high gene transfection efficiency, we synthesized a cationic polymer composed of low molecular weight polyethylenimine (PEI) of molecular weight of 600 Da cross-linked by 2-hydroxypropyl-gamma-cyclodextrin (HP gamma-CD) and then coupled to MC-10 oligopeptide containing a sequence of Met-Ala-Arg-Ala-Lys-Glu. The oligopeptide can target to HER2, the human epidermal growth factor receptor 2, which is often over expressed in many breast and ovary cancers. The new gene vector was expected to be able to target delivery of genes to HER2 positive cancer cells for gene therapy. The new gene vector was composed of chemically bonded HP gamma-CD, PEI (600 Da), and MC-10 peptide at a molar ratio of 1:3.3:1.2. The gene vector could condense plasmid DNA at an N/P ratio of 6 or above. The particle size of HP gamma-CD-PEI-P/DNA complexes at N/P ratios 40 was around 170-200 nm, with zeta potential of about 20 mV. The gene vector showed very low cytotoxicity, strong targeting specificity to HER2 receptor, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. The delivery of therapeutic IFN-alpha gene mediated by the new gene vector and the therapeutic efficiency were also studied in mice animal model. The animal study results showed that the new gene vector HP gamma-CD-PEI-P significantly enhanced the anti-tumor effect on tumor-bearing nude mice as compared to PEI (25 kDa), HP gamma-CD-PEI, and other controls, indicating that this new polymeric gene vector is a potential candidate for cancer gene therapy. (c) 2009 Elsevier Ltd. All rights reserved.

Zn(II) and Hg(II) binding to a designed peptide that accommodates different coordination geometries.

PubMed

Szunyogh, Dániel; Gyurcsik, Béla; Larsen, Flemming H; Stachura, Monika; Thulstrup, Peter W; Hemmingsen, Lars; Jancsó, Attila

2015-07-28

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both Zn(II) and Hg(II), i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, (1)H-NMR-, and PAC-spectroscopy as well as potentiometry. Hg(II) binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, Hg(II) is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in (1)H-NMR. The Zn(II)-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-Zn(II)-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications.

Identification, Characterization, and Recombinant Expression of Epidermicin NI01, a Novel Unmodified Bacteriocin Produced by Staphylococcus epidermidis That Displays Potent Activity against Staphylococci

PubMed Central

Sandiford, Stephanie

2012-01-01

We describe the discovery, purification, characterization, and expression of an antimicrobial peptide, epidermicin NI01, which is an unmodified bacteriocin produced by Staphylococcus epidermidis strain 224. It is a highly cationic, hydrophobic, plasmid-encoded peptide that exhibits potent antimicrobial activity toward a wide range of pathogenic Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA), enterococci, and biofilm-forming S. epidermidis strains. Purification of the peptide was achieved using a combination of hydrophobic interaction, cation exchange, and high-performance liquid chromatography (HPLC). Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis yielded a molecular mass of 6,074 Da, and partial sequence data of the peptide were elucidated using a combination of tandem mass spectrometry (MS/MS) and de novo sequencing. The draft genome sequence of the producing strain was obtained using 454 pyrosequencing technology, thus enabling the identification of the structural gene using the de novo peptide sequence data previously obtained. Epidermicin NI01 contains 51 residues with four tryptophan and nine lysine residues, and the sequence showed approximately 50% identity to peptides lacticin Z, lacticin Q, and aureocin A53, all of which belong to a new family of unmodified type II-like bacteriocins. The peptide is active in the nanomolar range against S. epidermidis, MRSA isolates, and vancomycin-resistant enterococci. Other unique features displayed by epidermicin include a high degree of protease stability and the ability to retain antimicrobial activity over a pH range of 2 to 10, and exposure to the peptide does not result in development of resistance in susceptible isolates. In this study we also show the structural gene alone can be cloned into Escherichia coli strain BL21(DE3), and expression yields active peptide. PMID:22155816

Cell Penetrating Peptides and Cationic Antibacterial Peptides

PubMed Central

Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

2014-01-01

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

[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.

The TV Turtle: A LOGO Graphics System for Raster Displays. AI Memo 361.

ERIC Educational Resources Information Center

Lieberman, Henry

This discussion of the advantages and limitations of raster graphics systems points out that until recently, most computer graphics systems have been oriented toward the display of line drawings, continually refreshing the screen from a display list of vectors. Developments such as plasma panel displays and rapidly declining memory prices have now…

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

PubMed

Takakusagi, Yoichi; Kuramochi, Kouji; Takagi, Manami; Kusayanagi, Tomoe; Manita, Daisuke; Ozawa, Hiroko; Iwakiri, Kanako; Takakusagi, Kaori; Miyano, Yuka; Nakazaki, Atsuo; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo

2008-11-15

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

PubMed

Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

PubMed Central

Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

Synergy of SOCS-1 Inhibition and Microbial-Based Cancer Vaccines

DTIC Science & Technology

2014-11-01

response without causing additional risk to the patient. The goal of our proposal is to modify a live- attenuated vaccine vector based on the food -borne...response after vaccination with a live-­‐‑attenuated L. monocytogenes. Aim 3: Test the hypothesis that secretion of a SOCS-­‐‑1 small peptide ...efficient internalization of pathogens and dying cells, processing of this material into peptide antigens that are presented in the context of major

Enhanced Peptide of Prostate Cancer Using Targeted Adenoviral Vectors

DTIC Science & Technology

2005-06-01

receptor subtype 2 has been constructed and evaluated in-human prostate cancer cells with regard to binding: of 64Cu - octreotide. In vivo experiments...of 64CU -octreotide.. The mice wer.e. sacrificed 1. h after peptide injection for biodistribution analysis. In vivo biodistribution studies showed...similar uptake of 64Cu - octreotide in both DU-145 and PC-3 tumors after infection with-AdSSTR2. (2.5. and 2.7% ID/g, respectively). This uptake was

CAPA-Gene Products in the Haematophagous Sandfly Phlebotomus papatasi (Scopoli) - Vector for Leishmaniasis Disease

DTIC Science & Technology

2012-01-01

121 glass capillary and the tissues were air-dried. For peptide analysis, 122 a limited amount of matrix solution (-cyano-4-hydroxycinnamic 123 acid ...genomic sequence of P. papatasi was screened with 143 the amino acid sequence RSGNMGLFPFPRVGR using TBLASTN. 144 The genomic data were produced by The...250 have the N-terminus of CAPA-PVK-2 blocked by pyroglutamate 251 (see Table 1). Pyroglutamate may prevent rapid degradation of this 252 peptide

The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

PubMed

Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

2014-03-01

Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to deliver neuroprotective drugs to the CNS following injury and/or potential neuroprotectants in their own right.

Multicomponent DNA carrier with a vesicular stomatitis virus G-peptide greatly enhances liver-targeted gene expression in mice.

PubMed

Schuster, M J; Wu, G Y; Walton, C M; Wu, C H

1999-01-01

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform.

PubMed

Mattiacio, Jonelle L; Brewer, Matt; Dewhurst, Stephen

2017-01-01

The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda phage capsid. Phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform.

Biological activity of Tat (47-58) peptide on human pathogenic fungi

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

Jung, Hyun Jun; Park, Yoonkyung; Department of Biotechnology, Chosun University, 375 Seosuk-dong, Kwangju 501-750

2006-06-23

Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol;more » 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.« less

Biological activity of Tat (47-58) peptide on human pathogenic fungi.

PubMed

Jung, Hyun Jun; Park, Yoonkyung; Hahm, Kyung-Soo; Lee, Dong Gun

2006-06-23

Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.

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.

Antimicrobial peptides: a new class of antimalarial drugs?

PubMed Central

Vale, Nuno; Aguiar, Luísa; Gomes, Paula

2014-01-01

A range of antimicrobial peptides (AMP) exhibit activity on malaria parasites, Plasmodium spp., in their blood or mosquito stages, or both. These peptides include a diverse array of both natural and synthetic molecules varying greatly in size, charge, hydrophobicity, and secondary structure features. Along with an overview of relevant literature reports regarding AMP that display antiplasmodial activity, this review makes a few considerations about those molecules as a potential new class of antimalarial drugs. PMID:25566072

Biodiscovery of Aluminum Binding Peptides

DTIC Science & Technology

2013-08-01

et al., "Biomimetic synthesis and patterning of silver nanoparticles ," Nat. Mater. 1(3), 169-172 (2002). [5] Van Dorst, B., et al., "Phage display...34Sequestration of zinc oxide by fimbrial designer chelators," Appl. Environ. Microbiol. 66(1), 10-14 (2000). [26] Hnilova, M., et al., "Peptide-directed co...biomaterial synthesis . Peptides have been developed that bind to a variety of inorganic materials, including metals1-6, oxides7, 8, alloys9, metal salts10

Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+ APC and antigen-specific T cell responses in vitro.

PubMed

Gogesch, Patricia; Schülke, Stefan; Scheurer, Stephan; Mühlebach, Michael D; Waibler, Zoe

2018-05-28

The development of novel vaccination strategies is a persistent challenge to provide effective prophylactic treatments to encounter viral infections. In general, the physical conjugation of selected vaccine components, e.g. antigen and adjuvant, has been shown to enhance the immunogenicity and hence, can increase effectiveness of the vaccine. In our proof-of-concept study, we generated non-infectious, replication deficient Murine Leukemia Virus (MLV)-derived virus-like particles (VLPs) that physically link antigen and adjuvant in a modular fashion by co-displaying them on their surface. For this purpose, we selected the immunodominant peptides of the model antigen ovalbumin (OVA) and the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) as non-classical adjuvant. Our results show that murine GM-CSF displayed on MLV-VLPs mediates expansion and proliferation of CD11b + cells within murine bone marrow and total spleen cells. Moreover, we show increased immunogenicity of modular VLPs co-displaying OVA peptides and GM-CSF by their elevated capacity to induce OVA-specific T cell-activation and -proliferation within OT-I and OT-II splenocyte cultures. These enhanced effects were not achieved by using an equimolar mixture of VLPs displaying either OVA or GM-CSF. Taken together, OVA and GM-CSF co-displaying MLV-VLPs are able to target and expand antigen presenting cells which in turn results in enhanced antigen-specific T cell activation and proliferation in vitro. These data suggest MLV-VLPs to be an attractive platform to flexibly combine antigen and adjuvant for novel modular vaccination approaches. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

PubMed

Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

2014-05-01

To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

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

Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.

Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of keymore » binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.« less

Production of human antimicrobial peptide LL-37 in Escherichia coli using a thioredoxin-SUMO dual fusion system.

PubMed

Li, Yifeng

2013-02-01

LL-37 is a human antimicrobial peptide that has been shown to possess multiple functions in host defense. In this report, the peptide was expressed as a fusion with a thioredoxin-SUMO dual-tag. Upon SUMO protease mediated cleavage at the SUMO/peptide junction, LL-37 with its native N-terminus was generated. The released peptide was separated from the dual-tag and cleavage enzyme by size-exclusion chromatography. Mass spectrometry analysis proves that the recombinant peptide has a molecular weight as theoretically expected for its native form. The produced peptide displayed antimicrobial activity against Escherichia coli K-12. On average, 2.4 mg peptide was obtained from one liter of bacterial culture. Thus, the described approach provides an effective alternative for producing active recombinant LL-37 with its natural amino acid sequence in E. coli. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

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.

Charge-reversal Lipids, Peptide-based Lipids, and Nucleoside-based Lipids for Gene Delivery

PubMed Central

LaManna, Caroline M.; Lusic, Hrvoje; Camplo, Michel; McIntosh, Thomas J.; Barthélémy, Philippe; Grinstaff, Mark W.

2013-01-01

Conspectus Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy’s appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to a myriad of diseases by tailoring the treatment to a specific individual’s genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when utilizing non-viral vectors have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: 1) that releasing the nucleic acid from the carrier will increase gene transfection; 2) that utilizing biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery; and 3) that mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will give unique supramolecular assembles with high transfection efficiency. The results presented in this Account demonstrate that cellular uptake and transfection efficacy can be improved by engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate change of the lipid from positive to negative net charge during the transfection process improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, and aromatic) between the cationic headgroup and the hydrophobic chains, lipids can be tailored to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. Introduction of a peptide headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in-vitro successes we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have utilized in our research, in order to provide readers with the tools to characterize and engineer new vectors. PMID:22439686

Real time display Fourier-domain OCT using multi-thread parallel computing with data vectorization

NASA Astrophysics Data System (ADS)

Eom, Tae Joong; Kim, Hoon Seop; Kim, Chul Min; Lee, Yeung Lak; Choi, Eun-Seo

2011-03-01

We demonstrate a real-time display of processed OCT images using multi-thread parallel computing with a quad-core CPU of a personal computer. The data of each A-line are treated as one vector to maximize the data translation rate between the cores of the CPU and RAM stored image data. A display rate of 29.9 frames/sec for processed OCT data (4096 FFT-size x 500 A-scans) is achieved in our system using a wavelength swept source with 52-kHz swept frequency. The data processing times of the OCT image and a Doppler OCT image with a 4-time average are 23.8 msec and 91.4 msec.

The effects of the phyllolitorin analogue [desTrp3,Leu8]phyllolitorin on scratching induced by bombesin and related peptides in rats

PubMed Central

Johnson, Mark D.; Ko, Mei-Chuan; Choo, Kevin S.; Traynor, John R.; Mosberg, Henry I.; Naughton, Norah N.; Woods, James H.

2010-01-01

Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue wdesTrp3,Leu8]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu8]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 μg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism. PMID:10482814

Expression and characterization of the antimicrobial peptide ABP-dHC-cecropin A in the methylotrophic yeast Pichia pastoris.

PubMed

Sang, Ming; Wei, Hui; Zhang, Jiaxin; Wei, Zhiheng; Wu, Xiaolong; Chen, Yan; Zhuge, Qiang

2017-12-01

ABP-dHC-cecropin A is a linear cationic peptide that exhibits antimicrobial properties. To explore a new approach for expression of ABP-dHC-cecropin A using the methylotrophic yeast Pichia pastoris, we cloned the ABP-dHC-cecropin A gene into the vector pPICZαA. The SacI-linearized plasmid pPICZαA-ABP-dHC-cecropin A was then transformed into P. pastoris GS115 by electroporation. Expression was induced after a 96-h incubation with 0.5% methanol at 20 °C in a culture supplied with 2% casamino acids to avoid proteolysis. Under these conditions, approximately 48 mg of ABP-dHC-cecropin A was secreted into 1L (4 × 250-mL)of medium. Recombinant ABP-dHC-cecropin A was purified using size-exclusion chromatography, and 21 mg of pure active ABP-dHC-cecropin A was obtained from 1L (4 × 250-mL)of culture. Electrophoresis on 4-20% gradient gels indicated that recombinant ABP-dHC-cecropin A was secreted as a protein approximately 4 kDa in size. Recombinant ABP-dHC-cecropin A was successfully expressed, as the product displayed antibacterial and antifungal activities (based on an antibacterial assay, scanning electron microscopy, and antifungal assay) indistinguishable from those of the synthesized protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Agent-based method for distributed clustering of textual information

DOEpatents

Potok, Thomas E [Oak Ridge, TN; Reed, Joel W [Knoxville, TN; Elmore, Mark T [Oak Ridge, TN; Treadwell, Jim N [Louisville, TN

2010-09-28

A computer method and system for storing, retrieving and displaying information has a multiplexing agent (20) that calculates a new document vector (25) for a new document (21) to be added to the system and transmits the new document vector (25) to master cluster agents (22) and cluster agents (23) for evaluation. These agents (22, 23) perform the evaluation and return values upstream to the multiplexing agent (20) based on the similarity of the document to documents stored under their control. The multiplexing agent (20) then sends the document (21) and the document vector (25) to the master cluster agent (22), which then forwards it to a cluster agent (23) or creates a new cluster agent (23) to manage the document (21). The system also searches for stored documents according to a search query having at least one term and identifying the documents found in the search, and displays the documents in a clustering display (80) of similarity so as to indicate similarity of the documents to each other.

New perspectives for natural antimicrobial peptides: application as antinflammatory drugs in a murine model.

PubMed

Capparelli, Rosanna; De Chiara, Francesco; Nocerino, Nunzia; Montella, Rosa Chiara; Iannaccone, Marco; Fulgione, Andrea; Romanelli, Alessandra; Avitabile, Concetta; Blaiotta, Giuseppe; Capuano, Federico

2012-11-17

Antimicrobial peptides (AMPs) are an ancient group of defense molecules. AMPs are widely distributed in nature (being present in mammals, birds, amphibians, insects, plants, and microorganisms). They display bactericidal as well as immunomodulatory properties. The aim of this study was to investigate the antimicrobial and anti-inflammatory activities of a combination of two AMPs (temporin B and the royal jellein I) against Staphylococcus epidermidis. The temporin B (TB-KK) and the royal jelleins I, II, III chemically modified at the C terminal (RJI-C, RJII-C, RJIII-C), were tested for their activity against 10 different Staphylococcus epidermidis strains, alone and in combination. Of the three royal jelleins, RJI-C showed the highest activity. Moreover, the combination of RJI-C and TB-KK (MIX) displayed synergistic activity. In vitro, the MIX displayed low hemolytic activity, no NO2- production and the ability to curb the synthesis of the pro-inflammatory cytokines TNF-α and IFN-γ to the same extent as acetylsalicylic acid. In vivo, the MIX sterilized mice infected with Staphylococcus epidermidis in eleven days and inhibited the expression of genes encoding the prostaglandin-endoperoxide synthase 2 (COX-2) and CD64, two important parameters of inflammation. The study shows that the MIX - a combination of two naturally occurring peptides - displays both antimicrobial and anti-inflammatory activities.

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.

A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

PubMed Central

Lehto, Taavi; Simonson, Oscar E; Mäger, Imre; Ezzat, Kariem; Sork, Helena; Copolovici, Dana-Maria; Viola, Joana R; Zaghloul, Eman M; Lundin, Per; Moreno, Pedro MD; Mäe, Maarja; Oskolkov, Nikita; Suhorutšenko, Julia; Smith, CI Edvard; Andaloussi, Samir EL

2011-01-01

Finding suitable nonviral delivery vehicles for nucleic acid–based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice. PMID:21343913

Measurements of Solar Vector Magnetic Fields

NASA Technical Reports Server (NTRS)

Hagyard, M. J. (Editor)

1985-01-01

Various aspects of the measurement of solar magnetic fields are presented. The four major subdivisions of the study are: (1) theoretical understanding of solar vector magnetic fields; (3) techniques for interpretation of observational data; and (4) techniques for data display.

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.

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.

A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin Vector

DTIC Science & Technology

2016-09-01

minidystrophin gene (a gift from Dr Jeffrey Chamberlain at the University of Washington, Seattle, WA) and the bovine growth hormone polyadenylation...full-length micro-dystrophin protein. Dys-2 is a short peptide in the wild-type full-length dystrophin. It can be recognized by the Dys-2...muscle. In one approach, a muscle homing peptide is inserted on the surface of the capsid to facilitate the entry of AAV into muscle cells. In the

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

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities.

PubMed

Bruning, Marc; Kreplak, Laurent; Leopoldseder, Sonja; Müller, Shirley A; Ringler, Philippe; Duchesne, Laurence; Fernig, David G; Engel, Andreas; Ucurum-Fotiadis, Zöhre; Mayans, Olga

2010-11-10

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.

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.

Phage-displayed peptides selected for binding to Campylobacter jejuni are antimicrobial.

PubMed

Bishop-Hurley, Sharon L; Rea, Philippa J; McSweeney, Christopher S

2010-10-01

In developed countries, Campylobacter jejuni is a leading cause of zoonotic bacterial gastroenteritis in humans with chicken meat implicated as a source of infection. Campylobacter jejuni colonises the lower gastrointestinal tract of poultry and during processing is spread from the gastrointestinal tract onto the surface of dressed carcasses. Controlling or eliminating C.jejuni on-farm is considered to be one of the best strategies for reducing human infection. Molecules on the cell surface of C.jejuni interact with the host to facilitate its colonisation and persistence in the gastrointestinal tract of poultry. We used a subtractive phage-display protocol to affinity select for peptides binding to the cell surface of a poultry isolate of C.jejuni with the aim of finding peptides that could be used to control this microorganism in chickens. In total, 27 phage peptides, representing 11 unique clones, were found to inhibit the growth of C.jejuni by up to 99.9% in vitro. One clone was bactericidal, reducing the viability of C.jejuni by 87% in vitro. The phage peptides were highly specific. They completely inhibited the growth of two of the four poultry isolates of C.jejuni tested with no activity detected towards other Gram-negative and Gram-positive bacteria.

Bacteriophage-fused peptides for serodiagnosis of human strongyloidiasis.

PubMed

Feliciano, Nágilla Daliane; Ribeiro, Vanessa da Silva; Santos, Fabiana de Almeida Araújo; Fujimura, Patricia Tiemi; Gonzaga, Henrique Tomaz; Goulart, Luiz Ricardo; Costa-Cruz, Julia Maria

2014-01-01

Strongyloidiasis, a human intestinal infection caused by the nematode Strongyloides stercoralis, is frequently underdiagnosed and although its high prevalence is still a neglected parasitic disease because conventional diagnostic tests based on parasitological examination (presence of Strongyloides larvae in stool) are not sufficiently sensitive due to the low parasitic load and to the irregular larval output. There is an urgent need to improve diagnostic assays, especially for immunocompromised patients with high parasitic load as consequence of self-infection cycle, which can disseminate throughout the body, resulting in a potentially fatal hyperinfection syndrome often accompanied by sepsis or meningitis. We have performed Phage Display technology to select peptides that mimic S. stercoralis antigens, capable of detecting a humoral response in patients with strongyloidiasis. The peptides reactivity was investigated by Phage-ELISA through different panels of serum samples. We have successfully selected five peptides with significant immunoreactivity to circulating IgG from patients' sera with strongyloidiasis. The phage displayed peptides C9 and C10 presented the highest diagnostic potential (AUC>0.87) with excellent sensitivity (>85%) and good specificity (>77.5%), suggesting that some S. stercoralis antigens trigger systemic immune response. These novel antigens are interesting serum biomarkers for routine strongyloidiasis screenings due to the easy production and simple assay using Phage-ELISA. Such markers may also present a promising application for therapeutic monitoring.

Quantitative PET Imaging with Novel HER3-Targeted Peptides Selected by Phage Display to Predict Androgen-Independent Prostate Cancer Progression

DTIC Science & Technology

2017-12-01

peptide in tumors that was linearly correlated with HER3 levels. Biodistribution analysis revealed low off-target accumulation and rapid clearance...Internal Lab 15-22 Dr. Larimer 5 Stock) Subtask 2: Correlate changes in peptide uptake with protein expression and cell signaling changes ex vivo...signal for each individual tumor was plotted against its corresponding HER3 protein level, the TBR correlated linearly with the amount of protein

Enterocin X, a Novel Two-Peptide Bacteriocin from Enterococcus faecium KU-B5, Has an Antibacterial Spectrum Entirely Different from Those of Its Component Peptides▿

PubMed Central

Hu, Chih-Bo; Malaphan, Wanna; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-01-01

Enterocin X, composed of two antibacterial peptides (Xα and Xβ), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xα and Xβ display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known. PMID:20418437

Integration of Peptides into Organic Thin Film Transistor (OTFT)-based Printable Sensors

DTIC Science & Technology

2017-02-10

AFRL-AFOSR-JP-TR-2017-0009 Integration of Peptides into Organic Thin Film Transistor (OTFT)-based Printable Sensors Paul Dastoor UNIVERSITY OF...collection of information   if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ORGANIZATION . 1...Peptides into Organic Thin Film Transistor (OTFT)-based Printable Sensors 5a.  CONTRACT NUMBER 5b.  GRANT NUMBER FA2386-15-1-4002 5c.  PROGRAM ELEMENT

Generic Biocombinatorial Strategy to Select Tailor-Made Stabilizers for Sol-Gel Nanoparticle Synthesis.

PubMed

Hanßke, Felix; Kemnitz, Erhard; Börner, Hans G

2015-09-09

A generic route for the selection of nanoparticle stabilizers via biocombinatorial means of phage display peptide screening is presented, providing magnesium fluoride nanoparticle synthesis as example. Selected sequence-specific MgF2 binders are evaluated for their adsorption behavior. Peptide-polymer conjugates derived from the best binding peptide are used for the stabilization of MgF2 sol nanoparticles, yielding fully redispersable dry states and improoving processability significantly. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Designed Electroresponsive Biomaterials: Sequence-Controlled Behavior

DTIC Science & Technology

2010-06-29

protein of the M13 . Traditional phage and yeast display methodologies indicate that peptide sequences with high affinities for electrode materials...drug delivery. The original vision for this work was to employ combinatorial tools such as phage and yeast display under electrical selection pressure...and drug delivery. The original vision for this work was to employ combinatorial tools such as phage and yeast display under electrical selection

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

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.

Self-assembled Nanomaterials for Chemotherapeutic Applications

NASA Astrophysics Data System (ADS)

Shieh, Aileen

The self-assembly of short designed peptides into functional nanostructures is becoming a growing interest in a wide range of fields from optoelectronic devices to nanobiotechnology. In the medical field, self-assembled peptides have especially attracted attention with several of its attractive features for applications in drug delivery, tissue regeneration, biological engineering as well as cosmetic industry and also the antibiotics field. We here describe the self-assembly of peptide conjugated with organic chromophore to successfully deliver sequence independent micro RNAs into human non-small cell lung cancer cell lines. The nanofiber used as the delivery vehicle is completely non-toxic and biodegradable, and exhibit enhanced permeability effect for targeting malignant tumors. The transfection efficiency with nanofiber as the delivery vehicle is comparable to that of the commercially available RNAiMAX lipofectamine while the toxicity is significantly lower. We also conjugated the peptide sequence with camptothecin (CPT) and observed the self-assembly of nanotubes for chemotherapeutic applications. The peptide scaffold is non-toxic and biodegradable, and drug loading of CPT is high, which minimizes the issue of systemic toxicity caused by extensive burden from the elimination of drug carriers. In addition, the peptide assembly drastically increases the solubility and stability of CPT under physiological conditions in vitro, while active CPT is gradually released from the peptide chain under the slight acidic tumor cell environment. Cytotoxicity results on human colorectal cancer cells and non-small cell lung cancer cell lines display promising anti-cancer properties compared to the parental CPT drug, which cannot be used clinically due to its poor solubility and lack of stability in physiological conditions. Moreover, the peptide sequence conjugated with 5-fluorouracil formed a hydrogel with promising topical chemotherapeutic applications that also display increased stability and controlled release of the active drug in vitro.

Parvovirus-like particles as vaccine vectors.

PubMed

Casal, J I; Rueda, P; Hurtado, A

1999-09-01

A wide array of systems have been developed to improve "classic" vaccines. The use of small polypeptides able to elicit potent antibody and cytotoxic responses seems to have enormous potential in the design of safer vaccines. While peptide coupling to large soluble proteins such as keyhole limpet hemocyanin is the current method of choice for eliciting antibody responses and insertion in live viruses for cytotoxic T-lymphocyte responses, alternative cheaper and/or safer methods will clearly be required in the future. Virus-like particles constitute very immunogenic molecules that allow for covalent coupling of the epitopes of interest in a simple way. In this article, we detail the methodology employed for the preparation of efficient virus vectors as delivery systems. We used parvovirus as the model for the design of new vaccine vectors. Recently parvovirus-like particles have been engineered to express foreign polypeptides in certain positions, resulting in the production of large quantities of highly immunogenic peptides, and to induce strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses. We discuss the different alternatives and the necessary steps to carry out this process, placing special emphasis on the flow of decisions that need to be made during the project. Copyright 1999 Academic Press.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

PubMed

Dekhtiarenko, Iryna; Ratts, Robert B; Blatnik, Renata; Lee, Lian N; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D; Marandu, Thomas F; Hoppe, Stephanie; Ruzsics, Zsolt; Sonnemann, Julia K; Mansouri, Mandana; Meyer, Christine; Lemmermann, Niels A W; Holtappels, Rafaela; Arens, Ramon; Klenerman, Paul; Früh, Klaus; Reddehase, Matthias J; Riemer, Angelika B; Cicin-Sain, Luka

2016-12-01

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors

PubMed Central

Blatnik, Renata; Lee, Lian N.; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D.; Marandu, Thomas F.; Hoppe, Stephanie; Ruzsics, Zsolt; Sonnemann, Julia K.; Meyer, Christine; Holtappels, Rafaela; Arens, Ramon; Früh, Klaus; Reddehase, Matthias J.; Riemer, Angelika B.; Cicin-Sain, Luka

2016-01-01

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy. PMID:27977791

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.

Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates.

PubMed

Mudgil, Priti; Kamal, Hina; Yuen, Gan Chee; Maqsood, Sajid

2018-09-01

In-vitro inhibitory properties of peptides released from camel milk proteins against dipeptidyl peptidase-IV (DPP-IV), porcine pancreatic α-amylase (PPA), and porcine pancreatic lipase (PPL) were studied. Results revealed that upon hydrolysis by different enzymes, camel milk proteins displayed dramatic increase in inhibition of DPP-IV and PPL, but slight improvement in PPA inhibition was noticed. Peptide sequencing revealed a total of 20 and 3 peptides for A9 and B9 hydrolysates respectively, obtained the score of 0.8 or more on peptide ranker and were categorized as potential DPP-IV inhibitory peptides. KDLWDDFKGL in A9 and MPSKPPLL in B9 were identified as most potent PPA inhibitory peptide. For PPL inhibition only 7 and 2 peptides qualified as PPL inhibitory peptides from hydrolysates A9 and B9, respectively. The present study report for the first time PPA and PPL inhibitory and only second for DPP-IV inhibitory potential of protein hydrolysates from camel milk. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Protein expression vector and secretion signal peptide optimization to drive the production, secretion, and functional expression of the bacteriocin enterocin A in lactic acid bacteria.

PubMed

Borrero, Juan; Jiménez, Juan J; Gútiez, Loreto; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

2011-10-20

Replacement of the leader sequence (LS) of the bacteriocin enterocin A (LS(entA)) by the signal peptides (SP) of the protein Usp45 (SP(usp45)), and the bacteriocins enterocin P (SP(entP)), and hiracin JM79 (SP(hirJM79)) permits the production, secretion, and functional expression of EntA by different lactic acid bacteria (LAB). Chimeric genes encoding the SP(usp45), the SP(entP), and the SP(hirJM79) fused to mature EntA plus the EntA immunity genes (entA+entiA) were cloned into the expression vectors pNZ8048 and pMSP3545, under control of the inducible P(nisA) promoter, and in pMG36c, under control of the constitutive P(32) promoter. The amount, antimicrobial activity, and specific antimicrobial activity of the EntA produced by the recombinant Lactococcus lactis, Enterococcus faecium, E. faecalis, Lactobacillus sakei and Pediococcus acidilactici hosts varied depending on the signal peptide, the expression vector, and the host strain. However, the antimicrobial activity and the specific antimicrobial activity of the EntA produced by most of the LAB transformants was lower than expected from their production. The supernatants of the recombinant L. lactis NZ9000 (pNZUAI) and L. lactis NZ9000 (pNZHAI), overproducers of EntA, showed a 1.2- to 5.1-fold higher antimicrobial activity than that of the natural producer E. faecium T136 against different Listeria spp. Copyright © 2011 Elsevier B.V. All rights reserved.

Peptide Based Radiopharmaceuticals: Specific Construct Approach

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

Som, P; Rhodes, B A; Sharma, S S

1997-10-21

The objective of this project was to develop receptor based peptides for diagnostic imaging and therapy. A series of peptides related to cell adhesion molecules (CAM) and immune regulation were designed for radiolabeling with 99mTc and evaluated in animal models as potential diagnostic imaging agents for various disease conditions such as thrombus (clot), acute kidney failure, and inflection/inflammation imaging. The peptides for this project were designed by the industrial partner, Palatin Technologies, (formerly Rhomed, Inc.) using various peptide design approaches including a newly developed rational computer assisted drug design (CADD) approach termed MIDAS (Metal ion Induced Distinctive Array of Structures).more » In this approach, the biological function domain and the 99mTc complexing domain are fused together so that structurally these domains are indistinguishable. This approach allows construction of conformationally rigid metallo-peptide molecules (similar to cyclic peptides) that are metabolically stable in-vivo. All the newly designed peptides were screened in various in vitro receptor binding and functional assays to identify a lead compound. The lead compounds were formulated in a one-step 99mTc labeling kit form which were studied by BNL for detailed in-vivo imaging using various animals models of human disease. Two main peptides usingMIDAS approach evolved and were investigated: RGD peptide for acute renal failure and an immunomodulatory peptide derived from tuftsin (RMT-1) for infection/inflammation imaging. Various RGD based metallopeptides were designed, synthesized and assayed for their efficacy in inhibiting ADP-induced human platelet aggregation. Most of these peptides displayed biological activity in the 1-100 µM range. Based on previous work by others, RGD-I and RGD-II were evaluated in animal models of acute renal failure. These earlier studies showed that after acute ischemic injury the renal cortex displays RGD receptor with higher density. The results have indicated good diagnostic potential for their use in this clinical situation, as an imaging agent to diagnose ischemic renal injury and differentiate from other causes. Very promising results were obtained with newly developed tuftsin related metallopeptides. A number of these peptides displayed high potency (nM range) in imaging infection. Antagonists were successfully used to image experimentally induced abscesses in rodents. One of the antagonists, termed 99mTc-RMT-1, was evaluated in rabbits and dogs for its applicability as infection/inflammation imaging agent. Both in dog and rabbit infection/inflammation models 99mTc-RMT-1 could be used for rapid scintigraphic diagnosis. A very high and rapid uptake was observed in both soft tissue and bone infection providing a good target to background contrast. The agent also allowed distinction between bone fracture and osteomyelitis. All these results warrant human clinical trials with 99mTc-RMT-1 which may help replace hazardous ex-vivo WBC labeling procedures that are current clincial modality for imaging infection foci.« less

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.

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.

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.

Engineering of the function of diamond-like carbon binding peptides through structural design.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Singh, Vivek K; Mikkilä, Joona; Kostiainen, Mauri A; Koskinen, Jari; Linder, Markus B

2015-02-09

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

Divergent unprotected peptide macrocyclisation by palladium-mediated cysteine arylation.

PubMed

Rojas, Anthony J; Zhang, Chi; Vinogradova, Ekaterina V; Buchwald, Nathan H; Reilly, John; Pentelute, Bradley L; Buchwald, Stephen L

2017-06-01

Macrocyclic peptides are important therapeutic candidates due to their improved physicochemical properties in comparison to their linear counterparts. Here we detail a method for a divergent macrocyclisation of unprotected peptides by crosslinking two cysteine residues with bis-palladium organometallic reagents. These synthetic intermediates are prepared in a single step from commercially available aryl bis-halides. Two bioactive linear peptides with cysteine residues at i , i + 4 and i , i + 7 positions, respectively, were cyclised to introduce a diverse array of aryl and bi-aryl linkers. These two series of macrocyclic peptides displayed similar linker-dependent lipophilicity, phospholipid affinity, and unique volume of distributions. Additionally, one of the bioactive peptides showed target binding affinity that was predominantly affected by the length of the linker. Collectively, this divergent strategy allowed rapid and convenient access to various aryl linkers, enabling the systematic evaluation of the effect of appending unit on the medicinal properties of macrocyclic peptides.

Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.

PubMed

Astafieva, A A; Rogozhin, E A; Odintsova, T I; Khadeeva, N V; Grishin, E V; Egorov, Ts A

2012-08-01

Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

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.

Disabled infectious single cycle-herpes simplex virus (DISC-HSV) as a vector for immunogene therapy of cancer.

PubMed

Rees, Robert C; McArdle, Stephanie; Mian, Shahid; Li, Geng; Ahmad, Murrium; Parkinson, Richard; Ali, Selman A

2002-02-01

Disabled infectious single cycle-herpes simplex viruses (DISC-HSV) have been shown to be safe for use in humans and may be considered efficacious as vectors for immunogene therapy in cancer. Preclinical studies show that DISC-HSV is an efficient delivery system for cytokine genes and antigens. DISC-HSV infects a high proportion of cells, resulting in rapid gene expression for at least 72 h. The DISC-HSV-mGM-CSF vector, when inoculated into tumors, induces tumor regression in a high percentage of animals, concomitant with establishing a cytotoxic T-cell response, which is MHC class I restricted and directed against peptides of known tumor antigens. The inherent properties of DISC-HSV makes it a suitable vector for consideration in human immunogene therapy trials.

Novel Optical Metamaterials and Approaches for Fabrication

DTIC Science & Technology

2012-08-01

phage display , we have also identified peptides that bind with nanoparticles and glass substrates. This is a critical step in engineering M13 ...with 2-mercaptoethanol ............................................... 12 Figure 13: DNA sequence of the three rounds of phage display selection with...corresponding amino acids ................................................................................ 13 Figure 14: M13 Phage bound to silicon

Using Phage Display to Create Recombinant Antibodies.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

A variety of phage display technologies have been developed since the approach was first described for antibodies. The most widely used approaches incorporate antibody sequences into the minor coat protein pIII of the nonlytic filamentous phage fd or M13. Libraries of variable gene sequences, encoding either scFv or Fab fragments, are made by incorporating sequences into phagemid vectors. The phagemid is packaged into phage particles with the assistance of a helper phage to produce the antibody display phage. This protocol describes a method for creating a phagemid library. The multiple cloning site (MCS) of the pBluescript KS(-) phagemid vector is replaced by digestion with the restriction enzyme BssHII, followed by the insertion of four overlapping oligonucleotides to create a new MCS within the vector. Next, the 3' portion of gene III (from M13mp18) is amplified and combined with an antibody sequence using overlap extension PCR. This product is inserted into the phagemid vector to create pPDS. Two helper plasmids are also created from the modified pBluescript vector: pLINK provides the linker between the heavy and light chains, and pFABC provides the CH1 domain of the heavy chain. An antibody cDNA library is constructed from the RNA of interest and ligated into pPDS. The phagemid library is electroporated into Escherichia coli cells along with the VCS-M13 helper phage. © 2017 Cold Spring Harbor Laboratory Press.

RNA detection using peptide-inserted Renilla luciferase.

PubMed

Andou, Takashi; Endoh, Tamaki; Mie, Masayasu; Kobatake, Eiry

2009-01-01

A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100 pM) exceeds that of Fluc by approximately two orders of magnitude.

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.

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

Ternary Aligned Nanofibers of RGD Peptide-Displaying M13 Bacteriophage/PLGA/Graphene Oxide for Facilitated Myogenesis

PubMed Central

Shin, Yong Cheol; Kim, Chuntae; Song, Su-Jin; Jun, Seungwon; Kim, Chang-Seok; Hong, Suck Won; Hyon, Suong-Hyu; Han, Dong-Wook; Oh, Jin-Woo

2018-01-01

Recently, there have been tremendous efforts to develop the biofunctional scaffolds by incorporating various biochemical factors. In the present study, we fabricated poly(lactic-co-glycolic acid) (PLGA) nanofiber sheets decorated with graphene oxide (GO) and RGD peptide. The decoration of GO and RGD peptide was readily achieved by using RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and electrospinning. Furthermore, the aligned GO-decorated PLGA/RGD peptide (GO-PLGA/RGD) ternary nanofiber sheets were prepared by magnetic field-assisted electrospinning, and their potentials as bifunctional scaffolds for facilitating myogenesis were explored. We characterized the physicochemical and mechanical properties of the sheets by scanning electron microscopy, Raman spectroscopy, contact angle measurement, and tensile test. In addition, the C2C12 skeletal myoblasts were cultured on the aligned GO-PLGA/RGD nanofiber sheets, and their cellular behaviors, including initial attachment, proliferation and myogenic differentiation, were evaluated. Our results revealed that the GO-PLGA/RGD nanofiber sheets had suitable physicochemical and mechanical properties for supporting cell growth, and could significantly promote the spontaneous myogenic differentiation of C2C12 skeletal myoblasts. Moreover, it was revealed that the myogenic differentiation was further accelerated on the aligned GO-PLGA/RGD nanofiber sheets due to the synergistic effects of RGD peptide, GO and aligned nanofiber structure. Therefore, , it is suggested that the aligned GO-PLGA/RGD ternary nanofiber sheets are one of the most promising approaches for facilitating myogenesis and promoting skeletal tissue regeneration. PMID:29577018

Ternary Aligned Nanofibers of RGD Peptide-Displaying M13 Bacteriophage/PLGA/Graphene Oxide for Facilitated Myogenesis.

PubMed

Shin, Yong Cheol; Kim, Chuntae; Song, Su-Jin; Jun, Seungwon; Kim, Chang-Seok; Hong, Suck Won; Hyon, Suong-Hyu; Han, Dong-Wook; Oh, Jin-Woo

2018-01-01

Recently, there have been tremendous efforts to develop the biofunctional scaffolds by incorporating various biochemical factors. In the present study, we fabricated poly(lactic- co -glycolic acid) (PLGA) nanofiber sheets decorated with graphene oxide (GO) and RGD peptide. The decoration of GO and RGD peptide was readily achieved by using RGD peptide-displaying M13 bacteriophage (RGD-M13 phage) and electrospinning. Furthermore, the aligned GO-decorated PLGA/RGD peptide (GO-PLGA/RGD) ternary nanofiber sheets were prepared by magnetic field-assisted electrospinning, and their potentials as bifunctional scaffolds for facilitating myogenesis were explored. We characterized the physicochemical and mechanical properties of the sheets by scanning electron microscopy, Raman spectroscopy, contact angle measurement, and tensile test. In addition, the C2C12 skeletal myoblasts were cultured on the aligned GO-PLGA/RGD nanofiber sheets, and their cellular behaviors, including initial attachment, proliferation and myogenic differentiation, were evaluated. Our results revealed that the GO-PLGA/RGD nanofiber sheets had suitable physicochemical and mechanical properties for supporting cell growth, and could significantly promote the spontaneous myogenic differentiation of C2C12 skeletal myoblasts. Moreover, it was revealed that the myogenic differentiation was further accelerated on the aligned GO-PLGA/RGD nanofiber sheets due to the synergistic effects of RGD peptide, GO and aligned nanofiber structure. Therefore, , it is suggested that the aligned GO-PLGA/RGD ternary nanofiber sheets are one of the most promising approaches for facilitating myogenesis and promoting skeletal tissue regeneration.

Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3.

PubMed

Taylor, Karen; Clarke, David J; McCullough, Bryan; Chin, Wutharath; Seo, Emily; Yang, De; Oppenheim, Joost; Uhrin, Dusan; Govan, John R W; Campopiano, Dominic J; MacMillan, Derek; Barran, Perdita; Dorin, Julia R

2008-03-14

beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.

PubMed

Abbassi, Feten; Raja, Zahid; Oury, Bruno; Gazanion, Elodie; Piesse, Christophe; Sereno, Denis; Nicolas, Pierre; Foulon, Thierry; Ladram, Ali

2013-02-01

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Cellular vaccines in listeriosis: role of the Listeria antigen GAPDH.

PubMed

Calderón-González, Ricardo; Frande-Cabanes, Elisabet; Bronchalo-Vicente, Lucía; Lecea-Cuello, M Jesús; Pareja, Eduardo; Bosch-Martínez, Alexandre; Fanarraga, Mónica L; Yañez-Díaz, Sonsoles; Carrasco-Marín, Eugenio; Alvarez-Domínguez, Carmen

2014-01-01

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189-201 and LLO91-99 and the GAPDH peptide, GAPDH1-22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1-22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91-99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1-22-specific CD4(+) and CD8(+) immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4(+) and CD8(+) epitopes.

Cellular vaccines in listeriosis: role of the Listeria antigen GAPDH

PubMed Central

Calderón-González, Ricardo; Frande-Cabanes, Elisabet; Bronchalo-Vicente, Lucía; Lecea-Cuello, M. Jesús; Pareja, Eduardo; Bosch-Martínez, Alexandre; Fanarraga, Mónica L.; Yañez-Díaz, Sonsoles; Carrasco-Marín, Eugenio; Álvarez-Domínguez, Carmen

2014-01-01

The use of live Listeria-based vaccines carries serious difficulties when administrated to immunocompromised individuals. However, cellular carriers have the advantage of inducing multivalent innate immunity as well as cell-mediated immune responses, constituting novel and secure vaccine strategies in listeriosis. Here, we compare the protective efficacy of dendritic cells (DCs) and macrophages and their safety. We examined the immune response of these vaccine vectors using two Listeria antigens, listeriolysin O (LLO) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and several epitopes such as the LLO peptides, LLO189−201 and LLO91−99 and the GAPDH peptide, GAPDH1−22. We discarded macrophages as safe vaccine vectors because they show anti-Listeria protection but also high cytotoxicity. DCs loaded with GAPDH1−22 peptide conferred higher protection and security against listeriosis than the widely explored LLO91−99 peptide. Anti-Listeria protection was related to the changes in DC maturation caused by these epitopes, with high production of interleukin-12 as well as significant levels of other Th1 cytokines such as monocyte chemotactic protein-1, tumor necrosis factor-α, and interferon-γ, and with the induction of GAPDH1−22-specific CD4+ and CD8+ immune responses. This is believed to be the first study to explore the use of a novel GAPDH antigen as a potential DC-based vaccine candidate for listeriosis, whose efficiency appears to highlight the relevance of vaccine designs containing multiple CD4+ and CD8+ epitopes. PMID:24600592

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

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

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions[OPEN

PubMed Central

Tavormina, Patrizia; De Coninck, Barbara; Nikonorova, Natalia; De Smet, Ive; Cammue, Bruno P.A.

2015-01-01

Peptides fulfill a plethora of functions in plant growth, development, and stress responses. They act as key components of cell-to-cell communication, interfere with signaling and response pathways, or display antimicrobial activity. Strikingly, both the diversity and amount of plant peptides have been largely underestimated. Most characterized plant peptides to date acting as small signaling peptides or antimicrobial peptides are derived from nonfunctional precursor proteins. However, evidence is emerging on peptides derived from a functional protein, directly translated from small open reading frames (without the involvement of a precursor) or even encoded by primary transcripts of microRNAs. These novel types of peptides further add to the complexity of the plant peptidome, even though their number is still limited and functional characterization as well as translational evidence are often controversial. Here, we provide a comprehensive overview of the reported types of plant peptides, including their described functional and structural properties. We propose a novel, unifying peptide classification system to emphasize the enormous diversity in peptide synthesis and consequent complexity of the still expanding knowledge on the plant peptidome. PMID:26276833

Live Attenuated Salmonella Vaccines Displaying Regulated Delayed Lysis and Delayed Antigen Synthesis To Confer Protection against Mycobacterium tuberculosis

PubMed Central

Juárez-Rodríguez, María Dolores; Yang, Jiseon; Kader, Rebin; Alamuri, Praveen; Curtiss, Roy

2012-01-01

Live recombinant attenuated Salmonella vaccine (RASV) strains have great potential to induce protective immunity against Mycobacterium tuberculosis by delivering M. tuberculosis antigens. Recently, we reported that, in orally immunized mice, RASV strains delivering the M. tuberculosis early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10) antigens via the Salmonella type III secretion system (SopE amino-terminal region residues 1 to 80 with two copies of ESAT-6 and one copy of CFP-10 [SopENt80-E2C]) afforded protection against aerosol challenge with M. tuberculosis. Here, we constructed and evaluated an improved Salmonella vaccine against M. tuberculosis. We constructed translational fusions for the synthesis of two copies of ESAT-6 plus CFP-10 fused to the OmpC signal sequence (OmpCSS-E2C) and amino acids 44 to 338 of antigen 85A (Ag85A294) flanked by the signal sequence (SS) and C-terminal peptide (CT) of β-lactamase (BlaSS-Ag85A294-BlaCT) to enable delivery via the Salmonella type II secretion system. The genes expressing these proteins were cloned as an operon transcribed from Ptrc into isogenic Asd+/MurA+ pYA3681 lysis vector derivatives with different replication origins (pBR, p15A, pSC101), resulting in pYA4890, pYA4891, and pYA4892 for SopENt80-E2C/Ag85A294 synthesis and pYA4893 and pYA4894 for OmpCSS-E2C/Ag85A294 synthesis. Mice orally immunized with the RASV χ11021 strain engineered to display regulated delayed lysis and regulated delayed antigen synthesis in vivo and harboring pYA4891, pYA4893, or pYA4894 elicited significantly greater humoral and cellular immune responses, and the RASV χ11021 strain afforded a greater degree of protection against M. tuberculosis aerosol challenge in mice than RASVs harboring any other Asd+/MurA+ lysis plasmid and immunization with M. bovis BCG, demonstrating that RASV strains displaying regulated delayed lysis with delayed antigen synthesis resulted in highly immunogenic delivery vectors for oral vaccination against M. tuberculosis infection. PMID:22144485

Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo.

PubMed

Roma-Rodrigues, Catarina; Heuer-Jungemann, Amelie; Fernandes, Alexandra R; Kanaras, Antonios G; Baptista, Pedro V

2016-01-01

In this work, peptides designed to selectively interact with cellular receptors involved in the regulation of angiogenesis were anchored to oligo-ethylene glycol-capped gold nanoparticles (AuNPs) and used to evaluate the modulation of vascular development using an ex ovo chick chorioallantoic membrane assay. These nanoparticles alter the balance between naturally secreted pro- and antiangiogenic factors, under various biological conditions, without causing toxicity. Exposure of chorioallantoic membranes to AuNP-peptide activators of angiogenesis accelerated the formation of new arterioles when compared to scrambled peptide-coated nanoparticles. On the other hand, antiangiogenic AuNP-peptide conjugates were able to selectively inhibit angiogenesis in vivo. We demonstrated that AuNP vectorization is crucial for enhancing the effect of active peptides. Our data showed for the first time the effective control of activation or inhibition of blood vessel formation in chick embryo via AuNP-based formulations suitable for the selective modulation of angiogenesis, which is of paramount importance in applications where promotion of vascular growth is desirable (eg, wound healing) or ought to be contravened, as in cancer development.

Comparison of different signal peptides for secretion of heterologous proteins in fission yeast

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

Kjaerulff, Soren; Jensen, Martin Roland

2005-10-28

In the fission yeast Schizosaccharomyces pombe, there are relatively few signal peptides available and most reports of their activity have not been comparative. Using sequence information from the S. pombe genome database we have identified three putative signal peptides, designated Cpy, Amy and Dpp, and compared their ability to support secretion of green fluorescent protein (GFP). In the comparison we also included the two well-described secretion signals derived from the precursors of, respectively, the Saccharomyces cerevisiae {alpha}-factor and the S. pombe P-factor. The capability of the tested signal peptides to direct secretion of GFP varied greatly. The {alpha}-factor signal didmore » not confer secretion to GFP and all the produced GFP was trapped intracellular. In contrast, the Cpy signal peptide supported efficient secretion of GFP with yields approximating 10 mg/L. We also found that the use of an attenuated version of the S. cerevisiae URA3 marker substantially increases vector copy number and expression yield in fission yeast.« less

Optimization of culturing conditions of recombined Escherichia coli to produce umami octopeptide-containing protein.

PubMed

Zhang, Yin; Wei, Xiong; Lu, Zhou; Pan, Zhongli; Gou, Xinhua; Venkitasamy, Chandrasekar; Guo, Siya; Zhao, Liming

2017-07-15

Using synthesized peptides to verify the taste of natural peptides was probably the leading cause for tasting disputes regarding umami peptides. A novel method was developed to prepare the natural peptide which could be used to verify the taste of umami peptide. A controversial octopeptide was selected and gene engineering was used to structure its Escherichia coli. expressing vector. A response surface method was adopted to optimize the expression conditions of the recombinant protein. The results of SDS-PAGE for the recombinant protein indicated that the recombinant expression system was successfully structured. The fitting results of the response surface experiment showed that the OD 600 value was the key factor which influenced the expression of the recombinant protein. The optimal culturing process conditions predicted with the fitting model were an OD 600 value of 0.5, an IPTG concentration of 0.6mM, a culturing temperature of 28.75°C and a culturing time of 5h. Copyright © 2017 Elsevier Ltd. All rights reserved.