Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

PubMed Central

Encell, Lance P; Friedman Ohana, Rachel; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G; Los, Georgyi V; McDougall, Mark G; Zimprich, Chad; Karassina, Natasha; Learish, Randall D; Hurst, Robin; Hartnett, James; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Mendez, Jacqui; Benink, Hélène A; Murphy, Nancy; Daniels, Danette L; Slater, Michael R; Urh, Marjeta; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2012-01-01

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins. PMID:23248739

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

PubMed

Mooney, Jane T; Fredericks, Dale P; Christensen, Thorkild; Bruun Schiødt, Christine; Hearn, Milton T W

2015-07-01

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes. Copyright © 2015 John Wiley & Sons, Ltd.

Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin

PubMed Central

Gabe, Claire M.; Brookes, Steven J.; Kirkham, Jennifer

2017-01-01

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond. PMID:28670287

High-level expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein fusion tag.

PubMed

Han, Yingqian; Guo, Wanying; Su, Bingqian; Guo, Yujie; Wang, Jiang; Chu, Beibei; Yang, Guoyu

2018-02-01

Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale production. The use of genetically engineered affinity and solubility enhancing fusion proteins has increased greatly in recent years, and there now exists a considerable repertoire of these that can be used to enhance the expression, stability, solubility, folding, and purification of their fusion partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a truncated maltotriose-binding protein (MBP; consisting of residues 59-433) from Pyrococcus furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site for protein expression and purification in Escherichia coli. Various proteins tagged at the N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore, four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to assess the affinity of HE with immobilized Ni 2+ . Our results showed that HE-MBP(Pyr) represents an attractive fusion protein allowing high levels of soluble expression and purification of recombinant protein in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.

PubMed

Mayer, Daniel; Baginsky, Sacha; Schwemmle, Martin

2005-11-01

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

A comparative evaluation of software for the analysis of liquid chromatography-tandem mass spectrometry data from isotope coded affinity tag experiments.

PubMed

Moulder, Robert; Filén, Jan-Jonas; Salmi, Jussi; Katajamaa, Mikko; Nevalainen, Olli S; Oresic, Matej; Aittokallio, Tero; Lahesmaa, Riitta; Nyman, Tuula A

2005-07-01

The options available for processing quantitative data from isotope coded affinity tag (ICAT) experiments have mostly been confined to software specific to the instrument of acquisition. However, recent developments with data format conversion have subsequently increased such processing opportunities. In the present study, data sets from ICAT experiments, analysed with liquid chromatography/tandem mass spectrometry (MS/MS), using an Applied Biosystems QSTAR Pulsar quadrupole-TOF mass spectrometer, were processed in triplicate using separate mass spectrometry software packages. The programs Pro ICAT, Spectrum Mill and SEQUEST with XPRESS were employed. Attention was paid towards the extent of common identification and agreement of quantitative results, with additional interest in the flexibility and productivity of these programs. The comparisons were made with data from the analysis of a specifically prepared test mixture, nine proteins at a range of relative concentration ratios from 0.1 to 10 (light to heavy labelled forms), as a known control, and data selected from an ICAT study involving the measurement of cytokine induced protein expression in human lymphoblasts, as an applied example. Dissimilarities were detected in peptide identification that reflected how the associated scoring parameters favoured information from the MS/MS data sets. Accordingly, there were differences in the numbers of peptides and protein identifications, although from these it was apparent that both confirmatory and complementary information was present. In the quantitative results from the three programs, no statistically significant differences were observed.

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

PubMed Central

Vergis, James M.; Wiener, Michael C.

2011-01-01

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase, Factor Xa, Human Rhinovirus 3C Protease, SUMOstar, Tobacco Etch Virus Protease, and Thrombin) by use of a panel of ninety-four individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and Factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919

Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

PubMed

Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

2016-03-01

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

Reversible chemoselective tagging and functionalization of methionine containing peptides.

PubMed

Kramer, Jessica R; Deming, Timothy J

2013-06-07

Reagents were developed to allow chemoselective tagging of methionine residues in peptides and polypeptides, subsequent bioorthogonal functionalization of the tags, and cleavage of the tags when desired. This methodology can be used for triggered release of therapeutic peptides, or release of tagged protein digests from affinity columns.

A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger.

PubMed

Roth, Andreas H F J; Dersch, Petra

2010-03-01

A set of different integrative expression vectors for the intracellular production of recombinant proteins with or without affinity tag in Aspergillus niger was developed. Target genes can be expressed under the control of the highly efficient, constitutive pkiA promoter or the novel sucrose-inducible promoter of the beta-fructofuranosidase (sucA) gene of A. niger in the presence or absence of alternative carbon sources. All expression plasmids contain an identical multiple cloning sequence that allows parallel construction of N- or C-terminally His6- and StrepII-tagged versions of the target proteins. Production of two heterologous model proteins, the green fluorescence protein and the Thermobifida fusca hydrolase, proved the functionality of the vector system. Efficient production and easy detection of the target proteins as well as their fast purification by a one-step affinity chromatography, using the His6- or StrepII-tag sequence, was demonstrated.

Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

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

Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael

In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a smallmore » change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.« less

Batch affinity adsorption of His-tagged proteins with EDTA-based chitosan.

PubMed

Hua, Weiwei; Lou, Yimin; Xu, Weiyuan; Cheng, Zhixian; Gong, Xingwen; Huang, Jianying

2016-01-01

Affinity adsorption purification of hexahistidine-tagged (His-tagged) proteins using EDTA-chitosan-based adsorption was designed and carried out. Chitosan was elaborated with ethylenediaminetetraacetic acid (EDTA), and the resulting polymer was characterized by FTIR, TGA, and TEM. Different metals including Ni(2+), Cu(2+), and Zn(2+) were immobilized with EDTA-chitosan, and their capability to the specific adsorption of His-tagged proteins were then investigated. The results showed that Ni(2+)-EDTA-chitosan and Zn(2+)-EDTA-chitosan had high affinity toward the His-tagged proteins, thus isolating them from protein mixture. The target fluorescent-labeled hexahistidine protein remained its fluorescent characteristic throughout the purification procedure when Zn(2+)-EDTA-chitosan was used as a sorbent, wherein the real-time monitor was performed to examine the immigration of fluorescent-labeled His-tagged protein. Comparatively, Zn(2+)-EDTA-chitosan showed more specific binding ability for the target protein, but with less binding capacity. It was further proved that this purification system could be recovered and reused at least for 5 times and could run on large scales. The presented M(2+)-EDTA-chitosan system, with the capability to specifically bind His-tagged proteins, make the purification of His-tagged proteins easy to handle, leaving out fussy preliminary treatment, and with the possibility of continuous processing and a reduction in operational cost in relation to the costs of conventional processes.

Optimal use of tandem biotin and V5 tags in ChIP assays

PubMed Central

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications.

PubMed

Young, Carissa L; Britton, Zachary T; Robinson, Anne S

2012-05-01

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology.

PubMed

Peccerella, Teresa; Lukan, Nadine; Hofheinz, Ralf; Schadendorf, Dirk; Kostrezewa, Markus; Neumaier, Michael; Findeisen, Peter

2010-02-01

The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant." Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

PubMed

Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

2014-11-01

Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.

GSyellow, a Multifaceted Tag for Functional Protein Analysis in Monocot and Dicot Plants.

PubMed

Besbrugge, Nienke; Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; Kulkarni, Shubhada R; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Bontinck, Michiel; Aesaert, Stijn; Impens, Francis; Gevaert, Kris; Van Damme, Daniel; Van Lijsebettens, Mieke; Inzé, Dirk; Vandepoele, Klaas; Nelissen, Hilde; De Jaeger, Geert

2018-06-01

The ability to tag proteins has boosted the emergence of generic molecular methods for protein functional analysis. Fluorescent protein tags are used to visualize protein localization, and affinity tags enable the mapping of molecular interactions by, for example, tandem affinity purification or chromatin immunoprecipitation. To apply these widely used molecular techniques on a single transgenic plant line, we developed a multifunctional tandem affinity purification tag, named GS yellow , which combines the streptavidin-binding peptide tag with citrine yellow fluorescent protein. We demonstrated the versatility of the GS yellow tag in the dicot Arabidopsis ( Arabidopsis thaliana ) using a set of benchmark proteins. For proof of concept in monocots, we assessed the localization and dynamic interaction profile of the leaf growth regulator ANGUSTIFOLIA3 (AN3), fused to the GS yellow tag, along the growth zone of the maize ( Zea mays ) leaf. To further explore the function of ZmAN3, we mapped its DNA-binding landscape in the growth zone of the maize leaf through chromatin immunoprecipitation sequencing. Comparison with AN3 target genes mapped in the developing maize tassel or in Arabidopsis cell cultures revealed strong conservation of AN3 target genes between different maize tissues and across monocots and dicots, respectively. In conclusion, the GS yellow tag offers a powerful molecular tool for distinct types of protein functional analyses in dicots and monocots. As this approach involves transforming a single construct, it is likely to accelerate both basic and translational plant research. © 2018 American Society of Plant Biologists. All rights reserved.

Pseudo-orthogonal frequency coded wireless SAW RFID temperature sensor tags.

PubMed

Saldanha, Nancy; Malocha, Donald C

2012-08-01

SAW sensors are ideal for various wireless, passive multi-sensor applications because they are small, rugged, radiation hard, and offer a wide range of material choices for operation over broad temperature ranges. The readable distance of a tag in a multi-sensor environment is dependent on the insertion loss of the device and the processing gain of the system. Single-frequency code division multiple access (CDMA) tags that are used in high-volume commercial applications must have universal coding schemes and large numbers of codes. The use of a large number of bits at the common center frequency to achieve sufficient code diversity in CDMA tags necessitates reflector banks with >30 dB loss. Orthogonal frequency coding is a spread-spectrum approach that employs frequency and time diversity to achieve enhanced tag properties. The use of orthogonal frequency coded (OFC) SAW tags reduces adjacent reflector interactions for low insertion loss, increased range, complex coding, and system processing gain. This work describes a SAW tag-sensor platform that reduces device loss by implementing long reflector banks with optimized spectral coding. This new pseudo-OFC (POFC) coding is defined and contrasted with the previously defined OFC coding scheme. Auto- and cross-correlation properties of the chips and their relation to reflectivity per strip and reflector length are discussed. Results at 250 MHz of 8-chip OFC and POFC SAW tags will be compared. The key parameters of insertion loss, cross-correlation, and autocorrelation of the two types of frequency-coded tags will be analyzed, contrasted, and discussed. It is shown that coded reflector banks can be achieved with near-zero loss and still maintain good coding properties. Experimental results and results predicted by the coupling of modes model are presented for varying reflector designs and codes. A prototype 915-MHz POFC sensor tag is used as a wireless temperature sensor and the results are shown.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: Insights into their use as tools for the removal of affinity tags

PubMed Central

Austin, Brian P.; Tözsér, József; Bagossi, Péter; Tropea, Joseph E.; Waugh, David S.

2012-01-01

Carboxypeptidases may serve as tools for removal for C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint. PMID:21073956

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor

PubMed Central

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G.M.

2017-01-01

Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. Here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker. The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. PMID:27867058

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

HaloTag Technology: A Versatile Platform for Biomedical Applications

PubMed Central

2015-01-01

Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications. PMID:25974629

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

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

Tykvart, J.; Sacha, P.; Barinka, C.

2012-02-07

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo.more » We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.« less

Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis.

PubMed

Kim, Young Hwan; Cho, Kun; Yun, Sung-Ho; Kim, Jin Young; Kwon, Kyung-Hoon; Yoo, Jong Shin; Kim, Seung Il

2006-02-01

Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jiménez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.

Construction of a chimeric thermostable pyrophosphatase to facilitate its purification and immobilization by using the choline-binding tag.

PubMed

Moldes, Cristina; García, José L; García, Pedro

2004-08-01

The thermophilic inorganic pyrophosphatase (Pyr) from Thermus thermophilus has been produced in Escherichia coli fused to the C terminus of the choline-binding tag (ChB tag) derived from the choline-binding domain (ChBD) of pneumococcal LytA autolysin. The chimeric ChBD-Pyr protein retains its thermostable activity and can be purified in a single step by DEAE-cellulose affinity chromatography. Pyr can be further released from the ChBD by thrombin, using the specific protease recognition site incorporated in the C terminus of this tag. Remarkably, the ChB tag provides a selective and very strong thermostable noncovalent immobilization of ChBD-Pyr in the DEAE-cellulose matrix. The binding of choline or choline analogues, such as DEAE, confers a high thermal stability to this tag; therefore, the immobilized chimeric enzyme can be assayed at high temperature without protein leakage, demonstrating the usefulness of the ChB tag for noncovalent immobilization of thermophilic proteins. Moreover, ChBD-Pyr can be purified and immobilized in a single step on commercial DEAE-cellulose paper. The affinity of the ChB tag for this versatile solid support can be very helpful in developing many biotechnological applications.

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Individually Identifiable Surface Acoustic Wave Sensors, Tags and Systems

NASA Technical Reports Server (NTRS)

Hines, Jacqueline H. (Inventor); Solie, Leland P. (Inventor); Tucker, Dana Y. G. (Inventor); Hines, Andrew T. (Inventor)

2017-01-01

A surface-launched acoustic wave sensor tag system for remotely sensing and/or providing identification information using sets of surface acoustic wave (SAW) sensor tag devices is characterized by acoustic wave device embodiments that include coding and other diversity techniques to produce groups of sensors that interact minimally, reducing or alleviating code collision problems typical of prior art coded SAW sensors and tags, and specific device embodiments of said coded SAW sensor tags and systems. These sensor/tag devices operate in a system which consists of one or more uniquely identifiable sensor/tag devices and a wireless interrogator. The sensor device incorporates an antenna for receiving incident RF energy and re-radiating the tag identification information and the sensor measured parameter(s). Since there is no power source in or connected to the sensor, it is a passive sensor. The device is wirelessly interrogated by the interrogator.

Scaffold design of trivalent chelator heads dictates high-affinity and stable His-tagged protein labeling in vitro and in cellulo.

PubMed

Gatterdam, Karl; Joest, Eike F; Gatterdam, Volker; Tampé, Robert

2018-05-29

Small chemical/biological interaction pairs are at the forefront in tracing proteins' function and interaction at high signal-to-background ratio in cellular pathways. Pharma ventures have eager plans to develop trisNTA probes for in vitro and in vivo screening of His-tagged protein targets. However, the optimal design of scaffold, linker, and chelator head yet deserves systematic investigations to achieve highest affinity and kinetic stability for in vitro and especially cell applications. In this study, we report on a library of N-nitrilotriacetic acid (NTA) based multivalent chelator heads (MCHs) built up on linear, cyclic, and dendritic scaffolds and contrast these with regard to their binding affinity and stability for labeling of cellular His-tagged proteins. Furthermore, we assign a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we describe fundamental differences between the MCH scaffold and define a cyclic trisNTA chelator, which displays the highest affinity and kinetic stability of all reversible, low-molecular weight interaction pairs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

Impact of an N-terminal Poly Histidine Tag on Protein Thermal Stability

USDA-ARS?s Scientific Manuscript database

For years, the use of polyhistidine tags (His-tags) have been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a cons...

Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein

PubMed Central

2013-01-01

Background In current protein research, a limitation still is the production of active recombinant proteins or native protein associations to assess their function. Especially the localization and analysis of protein-complexes or the identification of modifications and small molecule interaction partners by co-purification experiments requires a controllable expression of affinity- and/or fluorescence tagged variants of a protein of interest in its native cellular background. Advantages of periplasmic and/or homologous expressions can frequently not be realized due to a lack of suitable tools. Instead, experiments are often limited to the heterologous production in one of the few well established expression strains. Results Here, we introduce a series of new RK2 based broad host range expression plasmids for inducible production of affinity- and fluorescence tagged proteins in the cytoplasm and periplasm of a wide range of Gram negative hosts which are designed to match the recently suggested modular Standard European Vector Architecture and database. The vectors are equipped with a yellow fluorescent protein variant which is engineered to fold and brightly fluoresce in the bacterial periplasm following Sec-mediated export, as shown from fractionation and imaging studies. Expression of Strep-tag®II and Twin-Strep-tag® fusion proteins in Pseudomonas putida KT2440 is demonstrated for various ORFs. Conclusion The broad host range constructs we have produced enable good and controlled expression of affinity tagged protein variants for single-step purification and qualify for complex co-purification experiments. Periplasmic export variants enable production of affinity tagged proteins and generation of fusion proteins with a novel engineered Aequorea-based yellow fluorescent reporter protein variant with activity in the periplasm of the tested Gram-negative model bacteria Pseudomonas putida KT2440 and Escherichia coli K12 for production, localization or co-localization studies. In addition, the new tools facilitate metabolic engineering and yield assessment for cytoplasmic or periplasmic protein production in a number of different expression hosts when yields in one initially selected are insufficient. PMID:23687945

The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

PubMed

Amarasinghe, Chinthaka; Jin, Jian-Ping

2015-01-01

Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement.

Retention of coded wire tags, and their effect on maturation and survival of yellow mealworms (Coleoptera: Tenebrionidae)

USGS Publications Warehouse

Schaffler, James J.; Isely, J.J.

2001-01-01

This study demonstrates that coded wire tags can be used to mark certain insect larvae without adverse effects on maturation, and that tags are retained through the adult phase in high enough proportion for practical application. Coded wire tags also offer the benefit that marked organisms can be identified to the batch or individual level.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads. PMID:22688388

Purification of phage display-modified bacteriophage T4 by affinity chromatography

PubMed Central

2011-01-01

Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Structure-guided design of an engineered streptavidin with reusability to purify streptavidin-binding peptide tagged proteins or biotinylated proteins.

PubMed

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3-4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4 × 10(-14) M to 4.45 × 10(-10) M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

PubMed Central

Wu, Sau-Ching; Wong, Sui-Lam

2013-01-01

Development of a high-affinity streptavidin-binding peptide (SBP) tag allows the tagged recombinant proteins to be affinity purified using the streptavidin matrix without the need of biotinylation. The major limitation of this powerful technology is the requirement to use biotin to elute the SBP-tagged proteins from the streptavidin matrix. Tight biotin binding by streptavidin essentially allows the matrix to be used only once. To address this problem, differences in interactions of biotin and SBP with streptavidin were explored. Loop3–4 which serves as a mobile lid for the biotin binding pocket in streptavidin is in the closed state with biotin binding. In contrast, this loop is in the open state with SBP binding. Replacement of glycine-48 with a bulkier residue (threonine) in this loop selectively reduces the biotin binding affinity (Kd) from 4×10−14 M to 4.45×10−10 M without affecting the SBP binding affinity. Introduction of a second mutation (S27A) to the first mutein (G48T) results in the development of a novel engineered streptavidin SAVSBPM18 which could be recombinantly produced in the functional form from Bacillus subtilis via secretion. To form an intact binding pocket for tight binding of SBP, two diagonally oriented subunits in a tetrameric streptavidin are required. It is vital for SAVSBPM18 to be stably in the tetrameric state in solution. This was confirmed using an HPLC/Laser light scattering system. SAVSBPM18 retains high binding affinity to SBP but has reversible biotin binding capability. The SAVSBPM18 matrix can be applied to affinity purify SBP-tagged proteins or biotinylated molecules to homogeneity with high recovery in a reusable manner. A mild washing step is sufficient to regenerate the matrix which can be reused for multiple rounds. Other applications including development of automated protein purification systems, lab-on-a-chip micro-devices, reusable biosensors, bioreactors and microarrays, and strippable detection agents for various blots are possible. PMID:23874971

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

PubMed

Chen, Jia-Liang; Zhao, Yu; Gong, Yan-Jun; Pan, Bin-Bin; Wang, Xiao; Su, Xun-Cheng

2018-02-01

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

Evaluation and implementation of QR Code Identity Tag system for Healthcare in Turkey.

PubMed

Uzun, Vassilya; Bilgin, Sami

2016-01-01

For this study, we designed a QR Code Identity Tag system to integrate into the Turkish healthcare system. This system provides QR code-based medical identification alerts and an in-hospital patient identification system. Every member of the medical system is assigned a unique QR Code Tag; to facilitate medical identification alerts, the QR Code Identity Tag can be worn as a bracelet or necklace or carried as an ID card. Patients must always possess the QR Code Identity bracelets within hospital grounds. These QR code bracelets link to the QR Code Identity website, where detailed information is stored; a smartphone or standalone QR code scanner can be used to scan the code. The design of this system allows authorized personnel (e.g., paramedics, firefighters, or police) to access more detailed patient information than the average smartphone user: emergency service professionals are authorized to access patient medical histories to improve the accuracy of medical treatment. In Istanbul, we tested the self-designed system with 174 participants. To analyze the QR Code Identity Tag system's usability, the participants completed the System Usability Scale questionnaire after using the system.

Self-Powered Wireless Affinity-Based Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled RFID Antennas.

PubMed

Yuan, Mingquan; Alocilja, Evangelyn C; Chakrabartty, Shantanu

2016-08-01

This paper presents a wireless, self-powered, affinity-based biosensor based on the integration of paper-based microfluidics with our previously reported method for self-assembling radio-frequency (RF) antennas. At the core of the proposed approach is a silver-enhancement technique that grows portions of a RF antenna in regions where target antigens hybridize with target specific affinity probes. The hybridization regions are defined by a network of nitrocellulose based microfluidic channels which implement a self-powered approach to sample the reagent and control its flow and mixing. The integration substrate for the biosensor has been constructed using polyethylene and the patterning of the antenna on the substrate has been achieved using a low-cost ink-jet printing technique. The substrate has been integrated with passive radio-frequency identification (RFID) tags to demonstrate that the resulting sensor-tag can be used for continuous monitoring in a food supply-chain where direct measurement of analytes is typically considered to be impractical. We validate the proof-of-concept operation of the proposed sensor-tag using IgG as a model analyte and using a 915 MHz Ultra-high-frequency (UHF) RFID tagging technology.

Improved Tandem Affinity Purification Tag and Methods for Isolation of Proteins and Protein Complexes from Schizosaccharomyces pombe.

PubMed

Zilio, Nicola; Boddy, Michael N

2017-03-01

The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins. © 2017 Cold Spring Harbor Laboratory Press.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

PubMed

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2017-04-01

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli. Copyright © 2017 Elsevier Inc. All rights reserved.

The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.

PubMed

Imanaka, Hiroyuki; Yamadzumi, Daisuke; Yanagita, Keisuke; Ishida, Naoyuki; Nakanishi, Kazuhiro; Imamura, Koreyoshi

2016-03-01

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016. © 2016 American Institute of Chemical Engineers.

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

PubMed

Li, Junhua; Zhang, Yang; Yang, Yanjun

2013-03-01

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

A novel expression vector for the improved solubility of recombinant scorpion venom in Escherichia coli

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

Han, Tianqing; Ming, Hongyan; Deng, Lili

Recombinant scorpion anti-excitation peptide (rANEP) has previously been expressed using the pET32a system and purified via affinity chromatography. However, rANEP is expressed in BL21(DE3) cells as an inclusion body, and the affinity tag can not be removed. To overcome this problem, we used a variety of protein, DsbA, MBP, TrxA, intein, and affinity tags in fusion and co-expression to achieve soluble and functional rANEP without any affinity tag. In the pCIT-ANEP expression vector, the highest soluble expression level was approximately 90% of the total cellular proteins in E. coli, and the rANEP was cleaved by the intein protein and subsequently purifiedmore » to obtain rANEP, which had the same activity as the natural ANEP. The purity of rANEP obtained using this method was over 95%, with a quantity of 5.1 mg from of purified rANEP from 1 L of culture. This method could expand the application of the soluble expression of disulfide-rich peptides in E. coli.« less

Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

USDA-ARS?s Scientific Manuscript database

Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

Overview of the Purification of Recombinant Proteins

PubMed Central

Wingfield, Paul T.

2015-01-01

When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

Overview of the purification of recombinant proteins.

PubMed

Wingfield, Paul T

2015-04-01

When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

Application of Strep-Tactin XT for affinity purification of Twin-Strep-tagged CB2, a G protein-coupled cannabinoid receptor.

PubMed

Yeliseev, Alexei; Zoubak, Lioudmila; Schmidt, Thomas G M

2017-03-01

Human cannabinoid receptor CB 2 belongs to the class A of G protein-coupled receptor (GPCR). CB 2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB 2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB 2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB 2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB 2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB 2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB 2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies. Published by Elsevier Inc.

Bidirectional immobilization of affinity-tagged cytochrome c on electrode surfaces.

PubMed

Schröper, Florian; Baumann, Arnd; Offenhäusser, Andreas; Mayer, Dirk

2010-08-07

Here, we report a new strategy for the directed bivalent immobilization of cyt c on or between gold electrodes. C-terminal modification with cys- or his-tag did not affect the functional integrity of the protein. In combination with electrostatic protein binding, these tags enable a bifunctional immobilization between two electrodes or alternatively one electrode and interacting enzymes.

High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

PubMed Central

Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

2014-01-01

SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

PubMed Central

Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

2014-01-01

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

PubMed Central

Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

2012-01-01

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

PubMed

Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

2004-06-01

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

Review on SAW RFID tags.

PubMed

Plessky, Victor P; Reindl, Leonhard M

2010-03-01

SAW tags were invented more than 30 years ago, but only today are the conditions united for mass application of this technology. The devices in the 2.4-GHz ISM band can be routinely produced with optical lithography, high-resolution radar systems can be built up using highly sophisticated, but low-cost RF-chips, and the Internet is available for global access to the tag databases. The "Internet of Things," or I-o-T, will demand trillions of cheap tags and sensors. The SAW tags can overcome semiconductor-based analogs in many aspects: they can be read at a distance of a few meters with readers radiating power levels 2 to 3 orders lower, they are cheap, and they can operate in robust environments. Passive SAW tags are easily combined with sensors. Even the "anti-collision" problem (i.e., the simultaneous reading of many nearby tags) has adequate solutions for many practical applications. In this paper, we discuss the state-of-the-art in the development of SAW tags. The design approaches will be reviewed and optimal tag designs, as well as encoding methods, will be demonstrated. We discuss ways to reduce the size and cost of these devices. A few practical examples of tags using a time-position coding with 10(6) different codes will be demonstrated. Phase-coded devices can additionally increase the number of codes at the expense of a reduction of reading distance. We also discuss new and exciting perspectives of using ultra wide band (UWB) technology for SAW-tag systems. The wide frequency band available for this standard provides a great opportunity for SAW tags to be radically reduced in size to about 1 x 1 mm(2) while keeping a practically infinite number of possible different codes. Finally, the reader technology will be discussed, as well as detailed comparison made between SAW tags and IC-based semiconductor device.

Method of remote powering and detecting multiple UWB passive tags in an RFID system

DOEpatents

Dowla, Farid U [Castro Valley, CA; Nekoogar, Faranak [San Ramon, CA; Benzel, David M [Livermore, CA; Dallum, Gregory E [Livermore, CA; Spiridon, Alex [Palo Alto, CA

2012-05-29

A new Radio Frequency Identification (RFID), tracking, powering apparatus/system and method using coded Ultra-wideband (UWB) signaling is introduced. The proposed hardware and techniques disclosed herein utilize a plurality of passive UWB transponders in a field of an RFID-radar system. The radar system itself enables multiple passive tags to be remotely powered (activated) at about the same time frame via predetermined frequency UWB pulsed formats. Once such tags are in an activated state, an UWB radar transmits specific "interrogating codes" to put predetermined tags in an awakened status. Such predetermined tags can then communicate by a unique "response code" so as to be detected by an UWB system using radar methods.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

Barley as a green factory for the production of functional Flt3 ligand.

PubMed

Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

2010-02-01

Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

PubMed

Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

2015-09-01

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cellular Targets of Dietary Polyphenol Resveratrol

DTIC Science & Technology

2005-03-01

attempts to generate affinity columns tagged with other polyphenols, e.g., epigallocatechin gallate ( EGCG ). Conceivably such columns, if generated, would...Similar affinity chromatography with the related polyphenol Epigallocatechin gallate does not produce similar results.” Answer: We did not make...addition, the PI does not provid expression. If there is “increased ex many bind the resveratrol affinity co related polyphenol Epigallocatechin Response

High-throughput purification of recombinant proteins using self-cleaving intein tags.

PubMed

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

Tag retention, growth, and survival of red swamp crayfish Procambarus clarkii marked with coded wire tags

USGS Publications Warehouse

Isely, J.J.; Eversole, A.G.

1998-01-01

Juvenile red swamp crayfish (or crawfish), Procambarus clarkii (20-41 mm in total length) were collected from a crayfish culture pond by dipnetting and tagged with sequentially numbered, standard length, binary-coded wire tags. Four replicates of 50 crayfish were impaled perpendicular to the long axis of the abdomen with a fixed needle. Tags were injected transversely into the ventral surface of the first or second abdominal segment and were imbedded in the musculature just beneath the abdominal sternum. Tags were visible upon inspection. Additionally, two replicates of 50 crayfish were not tagged and were used as controls. Growth, survival, and tag retention were evaluated after 7 d in individual containers, after 100 d in aquaria, and after 200 d in field cages. Tag retention during each sample period was 100%, and average mortality of tagged crayfish within 7 d of tagging was 1%. Mortality during the remainder of the study was high (75-91%) but was similar between treatment and control samples. Most of the deaths were probably due to cannibalism. Average total length increased threefold during the course of the study, and crayfish reached maturity. Because crayfish were mature by the end of the study, we concluded that the coded wire tag was retained through the life history of the crayfish.

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

PubMed

Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

2016-03-15

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins.

PubMed

London, Anne Serdakowski; Patel, Kunal; Quinn, Lisa; Lemmerer, Martin

2015-04-01

Coupled affinity liquid chromatography and size exclusion chromatography (ALC-SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis. Copyright © 2014 Elsevier Inc. All rights reserved.

Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

PubMed

Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

2014-07-18

This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Z-path SAW RFID tag.

PubMed

Härmä, Sanna; Plessky, Victor P; Hartmann, Clinton S; Steichen, William

2008-01-01

Surface acoustic wave (SAW) radio-frequency identification (RFID) tags are soon expected to be produced in very high volumes. The size and cost of a SAW RFID tag will be key parameters for many applications. Therefore, it is of primary importance to reduce the chip size. In this work, we describe the design principles of a 2.4-GHz SAW RFID tag that is significantly smaller than earlier reported tags. We also present simulated and experimental results. The coded signal should arrive at the reader with a certain delay (typically about 1 micros), i.e., after the reception of environmental echoes. If the tag uses a bidirectional interdigital transducer (IDT), space for the initial delay is needed on both sides of the IDT. In this work, we replace the bidirectional IDT by a unidirectional one. This halves the space required by the initial delay because all the code reflectors must now be placed on the same side of the IDT. We reduce tag size even further by using a Z-path geometry in which the same space in x-direction is used for both the initial delay and the code reflectors. Chip length is thus determined only by the space required by the code reflectors.

Membrane protease degradomics: proteomic identification and quantification of cell surface protease substrates.

PubMed

Butler, Georgina S; Dean, Richard A; Smith, Derek; Overall, Christopher M

2009-01-01

The modification of cell surface proteins by plasma membrane and soluble proteases is important for physiological and pathological processes. Methods to identify shed and soluble substrates are crucial to further define the substrate repertoire, termed the substrate degradome, of individual proteases. Identifying protease substrates is essential to elucidate protease function and involvement in different homeostatic and disease pathways. This characterisation is also crucial for drug target identification and validation, which would then allow the rational design of specific targeted inhibitors for therapeutic intervention. We describe two methods for identifying and quantifying shed cell surface protease targets in cultured cells utilising Isotope-Coded Affinity Tags (ICAT) and Isobaric Tags for Relative and Absolute Quantification (iTRAQ). As a model system to develop these techniques, we chose a cell-membrane expressed matrix metalloproteinase, MMP-14, but the concepts can be applied to proteases of other classes. By over-expression, or conversely inhibition, of a particular protease with careful selection of control conditions (e.g. vector or inactive protease) and differential labelling, shed proteins can be identified and quantified by mass spectrometry (MS), MS/MS fragmentation and database searching.

Comparative analysis of viral RNA signatures on different RIG-I-like receptors

PubMed Central

Sanchez David, Raul Y; Combredet, Chantal; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Coppée, Jean-Yves; Mura, Marie; Guerbois Galla, Mathilde; Despres, Philippe; Tangy, Frédéric; Komarova, Anastassia V

2016-01-01

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection. DOI: http://dx.doi.org/10.7554/eLife.11275.001 PMID:27011352

DESIGN, SYNTHESIS, AND APPLICATION OF THE TRIMETHOPRIM-BASED CHEMICAL TAG FOR LIVE CELL IMAGING

PubMed Central

Jing, Chaoran; Cornish, Virginia W.

2013-01-01

Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live cell imaging. Alternative protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. PMID:23839994

Purification of bacteriophage lambda repressor

PubMed Central

Gao, Ning; Shearwin, Keith; Mack, John; Finzi, Laura; Dunlap, David

2013-01-01

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy “footprinting” assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops. PMID:23831434

[Identification of the interacting proteins with S100A8 or S100A9 by affinity purification and mass spectrometry].

PubMed

Wang, Jing; Zhang, Xuemei; Li, Zheng; Li, Xiayu; Ma, Jian; Shen, Shourong

2017-04-28

To identify the interacting proteins with S100A8 or S100A9 in HEK293 cell line by flag-tag affinity purification and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS).
 Methods: The p3×Flag-CMV-S100A8 and p3×Flag-CMV-S100A9 expression vectors were constructed by inserting S100A8 or S100A9 coding sequence. The recombinant plasmids were then transfected into HEK293 cells. Affinity purification and LC-MS/MS were applied to identify the proteins interacting with S100A8 or S100A9. Bioinformatics analysis was used to seek the gene ontology of the interacting proteins. Co-immunoprecipitation (Co-IP) was applied to confirm the proteins interacted with S100A8 or S100A9.
 Results: Fourteen proteins including pyruvate kinase, muscle (PKM), nucleophosmin (NPM1) and eukaryotic translation initiation factor 5A (EIF5A), which potentially interacted with S100A8, were successfully identified by Flag-tag affinity purification followed by LC-MS/MS analysis. Six proteins, such as tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (14-3-3ε) and PKM, which potentially interacted with S100A9, were successfully identified. Gene ontology analysis of the identified proteins suggested that proteins interacted with S100A8 or S100A9 were involved in several biological pathways, including canonical glycolysis, positive regulation of NF-κB transcription factor activity, negative regulation of apoptotic process, cell-cell adhesion, etc. Co-IP experiment confirmed that PKM2 can interact with both S100A8 and S100A9, and 14-3-3ε can interact with S100A8.
 Conclusion: PKM2 is identified to interact with both S100A8 and S100A9, while 14-3-3ε can interact with S100A9. These results may provide a new clue for the role of S100A8 or S100A9 in the progression of colitis-associated colorectal cancer.

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection

PubMed Central

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface. PMID:29360877

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

PubMed

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

Synthesis of a Polyhistidine-bearing Amphipol and its Use for Immobilizing Membrane Proteins.

PubMed

Giusti, Fabrice; Kessler, Pascal; Hansen, Randi Westh; Della Pia, Eduardo A; Le Bon, Christel; Mourier, Gilles; Popot, Jean-Luc; Martinez, Karen L; Zoonens, Manuela

2015-12-14

Amphipols (APols) are short amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions. In the present study, A8-35, a polyacrylate-based APol, was grafted with hexahistidine tags (His6-tags). The synthesis and characterization of this novel functionalized APol, named HistAPol, are described. Its ability to immobilize MPs on nickel ion-bearing surfaces was tested using two complementary methods, immobilized metal affinity chromatography (IMAC) and surface plasmon resonance (SPR). Compared to a single His6-tag fused at one extremity of a MP, the presence of several His6-tags carried by the APol belt surrounding the transmembrane domain of a MP increases remarkably the affinity of the protein/APol complex for nickel ion-bearing SPR chips, whereas it does not show such a strong effect on an IMAC resin. HistAPol-mediated immobilization, which allows reversibility of the interaction and easy regeneration of the supports and dispenses with any genetic modification of the target protein, provides a novel, promising tool for attaching MPs onto solid supports while stabilizing them.

Recovery of coded wire tags at a caspian tern colony in San Francisco Bay: A technique to evaluate impacts of avian predation on juvenile salmonids

USGS Publications Warehouse

Evans, A.F.; Roby, D.D.; Collis, K.; Cramer, B.M.; Sheggeby, J.A.; Adrean, L.J.; Battaglia, D.S.; Lyons, Donald E.

2011-01-01

We recovered coded wire tags (CWTs) from a colony of Caspian terns Hydroprogne caspia on Brooks Island in San Francisco Bay, California, to evaluate predation on juvenile salmonids originating from the Sacramento and San Joaquin rivers. Subsamples of colony substrate representing 11.7% of the nesting habitat used by the terns yielded 2,079 salmonid CWTs from fish released and subsequently consumed by terns in 2008. The estimated number of CWTs deposited on the entire tern colony was 40,143 (ranging from 26,763 to 80,288), once adjustments were made to account for tag loss and the total amount of nesting habitat used by terns. Tags ingested by terns and then egested on the colony were undamaged, and the tags' complete numeric codes were still identifiable. The CWTs found on the tern colony indicated that hatchery Chinook salmon Oncorhynchus tshawytscha trucked to and released in San Pablo Bay were significantly more likely to be consumed by Caspian terns than Chinook salmon that migrated in-river to the bay; 99.7% of all tags recovered were from bay-released Chinook salmon. Of the CWTs recovered on the tern colony, 98.0% were from fall-run Chinook salmon, indicating a higher susceptibility to tern predation than for the spring run type. None of the approximately 518,000 wild Chinook salmon that were coded-wire-tagged and released in the basin were recovered on the tern colony, suggesting that the impacts on wild, U.S. Endangered Species Act-listed Chinook salmon populations were minimal in 2008. Overall, we estimate that 0.3% of the approximately 12.3 million coded-wire-tagged Chinook salmon released in the basin in 2008 were subsequently consumed by Caspian terns from the Brooks Island colony. These results indicate that CWTs implanted in juvenile salmon can be recovered from a piscivorous waterbird colony and used to evaluate smolt losses for runs that are tagged. Abstract We recovered coded wire tags (CWTs) from a colony of Caspian terns Hydroprogne caspia on Brooks Island in San Francisco Bay, California, to evaluate predation on juvenile salmonids originating from the Sacramento and San Joaquin rivers. Subsamples of colony substrate representing 11.7% of the nesting habitat used by the terns yielded 2,079 salmonid CWTs from fish released and subsequently consumed by terns in 2008. The estimated number of CWTs deposited on the entire tern colony was 40,143 (ranging from 26,763 to 80,288), once adjustments were made to account for tag loss and the total amount of nesting habitat used by terns. Tags ingested by terns and then egested on the colony were undamaged, and the tags' complete numeric codes were still identifiable. The CWTs found on the tern colony indicated that hatchery Chinook salmon Oncorhynchus tshawytscha trucked to and released in San Pablo Bay were significantly more likely to be consumed by Caspian terns than Chinook salmon that migrated in-river to the bay; 99.7% of all tags recovered were from bay-released Chinook salmon. Of the CWTs recovered on the tern colony, 98.0% were from fall-run Chinook salmon, indicating a higher susceptibility to tern predation than for the spring run type. None of the approximately 518,000 wild Chinook salmon that were coded-wire-tagged and released in the basin were recovered on the tern colony, suggesting that the impacts on wild, U.S. Endangered Species Act-listed Chinook salmon populations were minimal in 2008. Overall, we estimate that 0.3% of the approximately 12.3 million coded-wire-tagged Chinook salmon released in the basin in 2008 were subsequently consumed by Caspian terns from the Brooks Island colony. These results indicate that CWTs implanted in juvenile salmon can be recovered from a piscivorous waterbird colony and used to evaluate smolt losses for runs that are tagged ?? American Fisheries Society 2011.

Effects of coded-wire-tagging on stream-dwelling Sea Lamprey larvae

USGS Publications Warehouse

Johnson, Nicholas; Swink, William D.; Dawson, Heather A.; Jones, Michael L.

2016-01-01

The effects of coded wire tagging Sea Lamprey Petromyzon marinus larvae from a known-aged stream-dwelling population were assessed. Tagged larvae were significantly shorter on average than untagged larvae from 3 to 18 months after tagging. However, 30 months after tagging, the length distribution of tagged and untagged larvae did not differ and tagged Sea Lampreys were in better condition (i.e., higher condition factor) and more likely to have undergone metamorphosis than the untagged population. The reason why tagged larvae were more likely to metamorphose is not clear, but the increased likelihood of metamorphosis could have been a compensatory response to the period of slower growth after tagging. Slower growth after tagging was consistent across larval size-classes, so handling and displacement from quality habitat during the early part of the growing season was likely the cause rather than the tag burden. The tag effects observed in this study, if caused by displacement and handling, may be minimized in future studies if tagging is conducted during autumn after growth has concluded for the year.

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

PubMed

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

The Design and Analysis of Salmonid Tagging Studies in the Columbia Basin; Volume XII; A Multinomial Model for Estimating Ocean Survival from Salmonid Coded Wire-Tag Data.

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

Ryding, Kristen E.; Skalski, John R.

1999-06-01

The purpose of this report is to illustrate the development of a stochastic model using coded wire-tag (CWT) release and age-at-return data, in order to regress first year ocean survival probabilities against coastal ocean conditions and climate covariates.

Weighted SAW reflector gratings for orthogonal frequency coded SAW tags and sensors

NASA Technical Reports Server (NTRS)

Puccio, Derek (Inventor); Malocha, Donald (Inventor)

2011-01-01

Weighted surface acoustic wave reflector gratings for coding identification tags and sensors to enable unique sensor operation and identification for a multi-sensor environment. In an embodiment, the weighted reflectors are variable while in another embodiment the reflector gratings are apodized. The weighting technique allows the designer to decrease reflectively and allows for more chips to be implemented in a device and, consequently, more coding diversity. As a result, more tags and sensors can be implemented using a given bandwidth when compared with uniform reflectors. Use of weighted reflector gratings with OFC makes various phase shifting schemes possible, such as in-phase and quadrature implementations of coded waveforms resulting in reduced device size and increased coding.

Summary of Findings from Coded Wire Tag Analysis from Spring Chinook Salmon Spawning Surveys in the Clearwater Basin, Technical Report 2001.

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

S.P. Cramer & Associates, Inc.

2002-05-31

We recently received data on the decoded coded wire tags (CWT's) recovered from spring chinook snouts we collected during spawning surveys in the Clearwater Basin last fall (2001). We were curious about what could be learned from the tags recovered (even though our project is over), so we did some cursory analyses and have described our findings in the attached memo. Snouts were processed and codes determined by Idaho Department of Fish and Game. Most snouts did not contain CWTs, because most ad-clipped fish were not given a CWT. Further, because adults were outplanted live, we do not know whatmore » codes they contained. Each of the hatcheries from which outplanted adults were obtained had several CWT code groups returning. That means that the best we can do with the codes recovered is compare the hatchery of origin for the tag with the hatchery from which outplants were taken. The results are interesting and not exactly as we would have predicted.« less

Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.

PubMed

Jing, Chaoran; Cornish, Virginia W

2013-01-01

Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included. © 2013 by John Wiley & Sons, Inc.

Error probability for RFID SAW tags with pulse position coding and peak-pulse detection.

PubMed

Shmaliy, Yuriy S; Plessky, Victor; Cerda-Villafaña, Gustavo; Ibarra-Manzano, Oscar

2012-11-01

This paper addresses the code reading error probability (EP) in radio-frequency identification (RFID) SAW tags with pulse position coding (PPC) and peak-pulse detection. EP is found in a most general form, assuming M groups of codes with N slots each and allowing individual SNRs in each slot. The basic case of zero signal in all off-pulses and equal signals in all on-pulses is investigated in detail. We show that if a SAW-tag with PPC is designed such that the spurious responses are attenuated by more than 20 dB below on-pulses, then EP can be achieved at the level of 10(-8) (one false read per 108 readings) with SNR >17 dB for any reasonable M and N. The tag reader range is estimated as a function of the transmitted power and EP.

Complete mitochondrial genome of Bactrocera arecae (Insecta: Tephritidae) by next-generation sequencing and molecular phylogeny of Dacini tribe

PubMed Central

Yong, Hoi-Sen; Song, Sze-Looi; Lim, Phaik-Eem; Chan, Kok-Gan; Chow, Wan-Loo; Eamsobhana, Praphathip

2015-01-01

The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general. PMID:26472633

78 FR 5797 - Availability of e-Tag Information to Commission Staff; Notice Specifying webRegistry Code

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-28

... e-Tag Information to Commission Staff; Notice Specifying webRegistry Code In Order No. 771,\\1\\ the... stated that, ``following issuance of this Final Rule and the Commission's registration in the OATI web... in the Purchasing-Seller Entity section of OATI webRegistry. This code should be used to designate...

Evidence for lack of homing by sea lampreys

USGS Publications Warehouse

Bergstedt, Roger A.; Seelye, James G.

1995-01-01

Recently metamorphosed sea lampreys Petromyzon marinus were captured in the Devil River, a tributary to Lake Huron, during summer and autumn 1990. They were tagged with a coded wire tag and returned to the river to continue their migration to Lake Huron to begin the parasitic (juvenile) phase of their life. During the spawning run in spring 1992 when the tagged animals were expected to mature and return to spawn, sea lampreys were trapped in nine tributaries to Lake Huron, including the Devil River; 47,946 animals were examined for coded wire tags, and 41 tagged animals were recovered. None of the 45 mature sea lampreys captured in the Devil River in 1992 were tagged, a proportion (0%) significantly lower than the proportion of the recently metamorphosed sea lampreys tagged in 1990. The distribution of tag recoveries among streams lakewide, however, was proportional to catch. Tagged sea lampreys did not appear to home, but instead seemed to select spawning streams through innate attraction to other sensory cues.

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

PubMed

Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

2016-05-01

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2016 Elsevier Inc. All rights reserved.

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.

PubMed

Mack, Laura; Brill, Boris; Delis, Natalia; Groner, Bernd

2014-12-01

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Identification on Membrane and Characterization of Phosphoproteins Using an Alkoxide-Bridged Dinuclear Metal Complex as a Phosphate-Binding Tag Molecule

PubMed Central

Nakanishi, Tsuyoshi; Ando, Eiji; Furuta, Masaru; Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru; Tsunasawa, Susumu; Nishimura, Osamu

2007-01-01

We have developed a method for on-membrane direct identification of phosphoproteins, which are detected by a phosphate-binding tag (Phos-tag) that has an affinity to phosphate groups with a chelated Zn2+ ion. This rapid profiling approach for phosphoproteins combines chemical inkjet technology for microdispensing of reagents onto a tiny region of target proteins with mass spectrometry for on-membrane digested peptides. Using this method, we analyzed human epidermoid carcinoma cell lysates of A-431 cells stimulated with epidermal growth factor, and identified six proteins with intense signals upon affinity staining with the phosphate-binding tag. It was already known that these proteins are phosphorylated, and our new approach proved to be effective at rapid profiling of phosphoproteins. Furthermore, we tried to determine their phosphorylation sites by MS/MS analysis after in-gel digestion of the corresponding spots on the 2DE gel to the rapid on-membrane identifications. As one example of use of information gained from the rapid-profiling approach, we successfully characterized a phosphorylation site at Ser-113 on prostaglandin E synthase 3. PMID:18166671

RNase One Gene Isolation, Expression, and Affinity Purification Models Research Experimental Progression and Culminates with Guided Inquiry-Based Experiments

ERIC Educational Resources Information Center

Bailey, Cheryl P.

2009-01-01

This new biochemistry laboratory course moves through a progression of experiments that generates a platform for guided inquiry-based experiments. RNase One gene is isolated from prokaryotic genomic DNA, expressed as a tagged protein, affinity purified, and tested for activity and substrate specificity. Student pairs present detailed explanations…

High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

PubMed

Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

2011-04-01

Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs.

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

PubMed

Ryan, Daniel P; Tremethick, David J

2016-04-01

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C. Copyright © 2015 Elsevier Inc. All rights reserved.

An internal affinity-tag for purification and crystallization of the siderophore receptor FhuA, integral outer membrane protein from Escherichia coli K-12.

PubMed

Ferguson, A D; Breed, J; Diederichs, K; Welte, W; Coulton, J W

1998-07-01

FhuA (Mr 78,992, 714 amino acids), siderophore receptor for ferrichrome-iron in the outer membrane of Escherichia coli, was affinity tagged, rapidly purified, and crystallized. To obtain FhuA in quantities sufficient for crystallization, a hexahistidine tag was genetically inserted into the fhuA gene after amino acid 405, which resides in a known surface-exposed loop. Recombinant FhuA405.H6 was overexpressed in an E. coli strain that is devoid of several major porins and using metal-chelate chromatography was purified in large amounts to homogeneity. FhuA crystals were grown using the hanging drop vapor diffusion technique and were suitable for X-ray diffraction analysis. On a rotating anode X-ray source, diffraction was observed to 3.0 A resolution. The crystals belong to space group P6(1) or P6(5) with unit cell dimensions of a=b=174 A, c=88 A (alpha=beta=90 degrees, gamma=120 degrees).

Annual Coded Wire Tag Program; Missing Production Groups, 1996 Annual Report.

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

Pastor, Stephen M.

1997-01-01

In 1989 the Bonneville Power Administration (BPA) began funding the evaluation of production groups of juvenile anadromous fish not being coded-wire tagged for other programs. These groups were the ''Missing Production Groups''. Production fish released by the U.S. Fish and Wildlife Service (USFWS) without representative coded-wire tags during the 1980's are indicated as blank spaces on the survival graphs in this report. The objectives of the ''Missing Production Groups'' program are: (1) to estimate the total survival of each production group, (2) to estimate the contribution of each production group to various fisheries, and (3) to prepare an annual reportmore » for all USFWS hatcheries in the Columbia River basin. Coded-wire tag recovery information will be used to evaluate the relative success of individual brood stocks. This information can also be used by salmon harvest managers to develop plans to allow the harvest of excess hatchery fish while protecting threatened, endangered, or other stocks of concern. In order to meet these objectives, a minimum of one marked group of fish is necessary for each production release. The level of marking varies according to location, species, and age at release. In general, 50,000 fish are marked with a coded-wire tag (CWT) to represent each production release group at hatcheries below John Day Dam. More than 100,000 fish per group are usually marked at hatcheries above John Day Dam. All fish release information, including marked/unmarked ratios, is reported to the Pacific States Marine Fisheries Commission (PSMFC). Fish recovered in the various fisheries or at the hatcheries are sampled to recover coded-wire tags. This recovery information is also reported to PSMFC.« less

The structure of the SBP-Tag–streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

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

Barrette-Ng, Isabelle H.; Wu, Sau-Ching; Tjia, Wai-Mui

2013-05-01

The structure of the SBP-Tag–streptavidin complex reveals a novel mode of peptide recognition in which a single peptide binds simultaneously to biotin-binding pockets from adjacent subunits of streptavidin. The molecular details of peptide recognition suggest how the SBP-Tag can be further modified to become an even more useful tag for a wider range of biotechnological applications. The 38-residue SBP-Tag binds to streptavidin more tightly (K{sub d} ≃ 2.5–4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-bindingmore » pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12–14) and a C-terminal HPQ sequence (residues 31–33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17–28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1–10 and 35–38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11–34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag–streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.« less

Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

PubMed Central

De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

2014-01-01

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation. PMID:24599037

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

PubMed

De Souza, Colin P; Hashmi, Shahr B; Osmani, Aysha H; Osmani, Stephen A

2014-01-01

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

PubMed

Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

2006-01-01

Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

76 FR 71315 - Endangered and Threatened Species; Take of Anadromous Fish

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-17

... the fish and would then sample them for biological information (fin tissue and scale samples). They..., measured, weighed, tissue-sampled, and checked for external marks and coded-wire tags depending on the.... Then the researchers would remove and preserve fish body tissues, otoliths, and coded wire tags (from...

Isolation of centromeric-tandem repetitive DNA sequences by chromatin affinity purification using a HaloTag7-fused centromere-specific histone H3 in tobacco.

PubMed

Nagaki, Kiyotaka; Shibata, Fukashi; Kanatani, Asaka; Kashihara, Kazunari; Murata, Minoru

2012-04-01

The centromere is a multi-functional complex comprising centromeric DNA and a number of proteins. To isolate unidentified centromeric DNA sequences, centromere-specific histone H3 variants (CENH3) and chromatin immunoprecipitation (ChIP) have been utilized in some plant species. However, anti-CENH3 antibody for ChIP must be raised in each species because of its species specificity. Production of the antibodies is time-consuming and costly, and it is not easy to produce ChIP-grade antibodies. In this study, we applied a HaloTag7-based chromatin affinity purification system to isolate centromeric DNA sequences in tobacco. This system required no specific antibody, and made it possible to apply a highly stringent wash to remove contaminated DNA. As a result, we succeeded in isolating five tandem repetitive DNA sequences in addition to the centromeric retrotransposons that were previously identified by ChIP. Three of the tandem repeats were centromere-specific sequences located on different chromosomes. These results confirm the validity of the HaloTag7-based chromatin affinity purification system as an alternative method to ChIP for isolating unknown centromeric DNA sequences. The discovery of more than two chromosome-specific centromeric DNA sequences indicates the mosaic structure of tobacco centromeres. © Springer-Verlag 2011

Host Immune Selection of Rumen Bacteria through Salivary Secretory IgA

PubMed Central

Fouhse, Janelle M.; Smiegielski, Luke; Tuplin, Melanie; Guan, Le Luo; Willing, Benjamin P.

2017-01-01

The rumen microbiome is integral to efficient production in cattle and shows strong host specificity, yet little is known about what host factors shape rumen microbial composition. Secretory immunoglobulin A (SIgA) is produced in large amounts in the saliva, can coat both commensal and pathogenic microbes within the gut, and presents a plausible mechanism of host specificity. However, the role salivary SIgA plays in commensal bacteria selection in ruminants remains elusive. The main objectives of this study were to develop an immuno-affinity benchtop method to isolate SIgA-tagged microbiota and to determine if salivary SIgA preferentially binds selected bacteria. We hypothesized that SIgA-tagged bacteria would differ from total bacteria, thus supporting a potential host-derived mechanism in commensal bacterial selection. Whole rumen (n = 9) and oral secretion samples (n = 10) were incubated with magnetic beads conjugated with anti-secretory IgA antibodies to enrich SIgA-tagged microbiota. Microbial DNA from the oral secretion, whole rumen, SIgA-tagged oral secretion, and SIgA-tagged rumen was isolated for amplicon sequencing of V1–V3 region of 16S rDNA genes. Whole rumen and oral secretion had distinctive (P < 0.05) bacterial compositions indicated by the non-parametric multidimensional scaling plot using Euclidean distance metrics. The SIgA-tagged microbiota from rumen and oral secretion had similar abundance of Bacteroidetes, Actinobacteria, Fibrobacter, candidate phyla TM7, and Tenericutes and are clustered tightly. Composition of SIgA-tagged oral secretion microbiota was more similar to whole rumen microbiota than whole oral secretion due to enrichment of rumen bacteria (Lachnospiraceae) and depletion of oral taxa (Streptococcus, Rothia, Neisseriaceae, and Lactobacillales). In conclusion, SIgA-tagged oral secretion microbiota had an increased resemblance to whole rumen microbiota, suggesting salivary SIgA-coating may be one host-derived mechanism impacting commensal colonization. Further studies, to explore the variations in antibody affinity between different animals as a driver of microbial composition are warranted. PMID:28553275

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

PubMed

Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

2006-10-01

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

PubMed

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2016-02-01

Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

Strep-Tagged Protein Purification.

PubMed

Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

2015-01-01

The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). © 2015 Elsevier Inc. All rights reserved.

Annual Stock Assessment - CWT [Coded Wire Tag program] (USFWS), Annual Report 2007.

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

Pastor, Stephen M.

2009-07-21

In 1989 the Bonneville Power Administration (BPA) began funding the evaluation of production groups of juvenile anadromous fish not being coded-wire tagged for other programs. These groups were the 'Missing Production Groups'. Production fish released by the U.S. Fish and Wildlife Service (FWS) without representative coded-wire tags during the 1980s are indicated as blank spaces on the survival graphs in this report. This program is now referred to as 'Annual Stock Assessment - CWT'. The objectives of the 'Annual Stock Assessment' program are to: (1) estimate the total survival of each production group, (2) estimate the contribution of each productionmore » group to fisheries, and (3) prepare an annual report for USFWS hatcheries in the Columbia River basin. Coded-wire tag recovery information will be used to evaluate the relative success of individual brood stocks. This information can also be used by salmon harvest managers to develop plans to allow the harvest of excess hatchery fish while protecting threatened, endangered, or other stocks of concern. All fish release information, including marked/unmarked ratios, is reported to the Pacific States Marine Fisheries Commission (PSMFC). Fish recovered in the various fisheries or at the hatcheries are sampled to recover coded-wire tags. This recovery information is also reported to PSMFC. This report has been prepared annually starting with the report labeled 'Annual Report 1994'. Although the current report has the title 'Annual Report 2007', it was written in fall of 2008 using data available from RMIS that same year, and submitted as final in January 2009. The main objective of the report is to evaluate survival of groups which have been tagged under this ongoing project.« less

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425

A Phos-tag-based magnetic-bead method for rapid and selective separation of phosphorylated biomolecules.

PubMed

Tsunehiro, Masaya; Meki, Yuma; Matsuoka, Kanako; Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Koike, Tohru

2013-04-15

A simple and efficient method based on magnetic-bead technology has been developed for the separation of phosphorylated and nonphosphorylated low-molecular-weight biomolecules, such as nucleotides, phosphorylated amino acids, or phosphopeptides. The phosphate-binding site on the bead is an alkoxide-bridged dinuclear zinc(II) complex with 1,3-bis(pyridin-2-ylmethylamino)propan-2-olate (Phos-tag), which is linked to a hydrophilic cross-linked agarose coating on a magnetic core particle. All steps for the phosphate-affinity separation are conducted in buffers of neutral pH with 50 μL of the magnetic beads in a 1.5-mL microtube. The entire separation protocol for phosphomonoester-type compounds, from addition to elution, requires less than 12 min per sample if the buffers and the zinc(II)-bound Phos-tag magnetic beads have been prepared in advance. The phosphate-affinity magnetic beads are reusable at least 15 times without a decrease in their phosphate-binding ability and they are stable for three months in propan-2-ol. Copyright © 2013 Elsevier B.V. All rights reserved.

System and method for a parallel immunoassay system

DOEpatents

Stevens, Fred J.

2002-01-01

A method and system for detecting a target antigen using massively parallel immunoassay technology. In this system, high affinity antibodies of the antigen are covalently linked to small beads or particles. The beads are exposed to a solution containing DNA-oligomer-mimics of the antigen. The mimics which are reactive with the covalently attached antibody or antibodies will bind to the appropriate antibody molecule on the bead. The particles or beads are then washed to remove any unbound DNA-oligomer-mimics and are then immobilized or trapped. The bead-antibody complexes are then exposed to a test solution which may contain the targeted antigens. If the antigen is present it will replace the mimic since it has a greater affinity for the respective antibody. The particles are then removed from the solution leaving a residual solution. This residual solution is applied a DNA chip containing many samples of complimentary DNA. If the DNA tag from a mimic binds with its complimentary DNA, it indicates the presence of the target antigen. A flourescent tag can be used to more easily identify the bound DNA tag.

Tagging as a Social Literacy Practice

ERIC Educational Resources Information Center

MacGillivray, Laurie; Curwen, Margaret Sauceda

2007-01-01

Tagging is not simply an act of vandalism or violence; it is a social practice with its own rules and codes--a literacy practice imbued with intent and meaning. Three aspects of tagging reflect its nature as a literate practice: (1) The purpose of tagging to achieve particular social goals and group affiliations; (2) The role of talent to be…

Identification coding schemes for modulated reflectance systems

DOEpatents

Coates, Don M [Santa Fe, NM; Briles, Scott D [Los Alamos, NM; Neagley, Daniel L [Albuquerque, NM; Platts, David [Santa Fe, NM; Clark, David D [Santa Fe, NM

2006-08-22

An identifying coding apparatus employing modulated reflectance technology involving a base station emitting a RF signal, with a tag, located remotely from the base station, and containing at least one antenna and predetermined other passive circuit components, receiving the RF signal and reflecting back to the base station a modulated signal indicative of characteristics related to the tag.

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

PubMed

Pesavento, James J; Bullock, Courtney R; LeDuc, Richard D; Mizzen, Craig A; Kelleher, Neil L

2008-05-30

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

Quantitative interaction proteomics using mass spectrometry.

PubMed

Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

2009-03-01

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

PubMed

Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

2015-12-01

To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

Summary of evidence for an anticodonic basis for the origin of the genetic code

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.

1981-01-01

This article summarizes data supporting the hypothesis that the genetic code origin was based on relationships (probably affinities) between amino acids and their anticodon nucleotides. Selective activation seems to follow from selective affinity and consequently, incorporation of amino acids into peptides can also be selective. It is suggested that these selectivities in affinity and activation, coupled with the base pairing specificities, allowed the origin of the code and the process of translation.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Using Digital Acoustic Recording Tags to Detect Marine Mammals on Navy Ranges and Study their Responses to Naval Sonar

DTIC Science & Technology

2011-02-01

written in C and assembly languages. 2) executable code for the low-power wakeup controller in the tag. This software is responsible for the VHF...used in the tag software. The multi-rate processing in the new tag necessitated a more complex task- scheduling software architecture. The effort of

Short range spread-spectrum radiolocation system and method

DOEpatents

Smith, Stephen F.

2003-04-29

A short range radiolocation system and associated methods that allow the location of an item, such as equipment, containers, pallets, vehicles, or personnel, within a defined area. A small, battery powered, self-contained tag is provided to an item to be located. The tag includes a spread-spectrum transmitter that transmits a spread-spectrum code and identification information. A plurality of receivers positioned about the area receive signals from a transmitting tag. The position of the tag, and hence the item, is located by triangulation. The system employs three different ranging techniques for providing coarse, intermediate, and fine spatial position resolution. Coarse positioning information is provided by use of direct-sequence code phase transmitted as a spread-spectrum signal. Intermediate positioning information is provided by the use of a difference signal transmitted with the direct-sequence spread-spectrum code. Fine positioning information is provided by use of carrier phase measurements. An algorithm is employed to combine the three data sets to provide accurate location measurements.

An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

PubMed

Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

2011-01-01

cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Retention of internal anchor tags by juvenile striped bass

USGS Publications Warehouse

Van Den Avyle, M.J.; Wallin, J.E.

2001-01-01

We marked hatchery-reared striped bass Morone saxatilis (145-265 mm total length) with internal anchor tags and monitored retention for 28 months after stocking in the Savannah River, Georgia and South Carolina. Anchor tags (with an 18-mm, T-shaped anchor and 42-mm streamer) were surgically implanted ventrally, and coded wire tags (1 mm long and 0.25 mm in diameter) were placed into the cheek muscle to help identify subsequent recaptures. The estimated probability of retention (SD) of anchor tags was 0.94 (0.05) at 4 months, 0.64 (0.13) at 16 months, and 0.33 (0.19) at 28 months. Of 10 fish recaptured with only coded wire tags, 5 showed an externally visible wound or scar near the point of anchor tag insertion. The incidence of wounds or scars, which we interpreted as evidence of tag shedding, increased to 50% in recaptures taken at 28 months (three of six fish). Our estimates for retention of anchor tags were generally lower than those in other studies of striped bass, possibly because of differences in the style of anchor or sizes of fish used. Because of its low rate of retention, the type of anchor tag we used may not be suitable for long-term assessments of stock enhancement programs that use striped bass of the sizes we evaluated.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

A General Strategy for Targeting Drugs to Bone.

PubMed

Jahnke, Wolfgang; Bold, Guido; Marzinzik, Andreas L; Ofner, Silvio; Pellé, Xavier; Cotesta, Simona; Bourgier, Emmanuelle; Lehmann, Sylvie; Henry, Chrystelle; Hemmig, René; Stauffer, Frédéric; Hartwieg, J Constanze D; Green, Jonathan R; Rondeau, Jean-Michel

2015-11-23

Targeting drugs to their desired site of action can increase their safety and efficacy. Bisphosphonates are prototypical examples of drugs targeted to bone. However, bisphosphonate bone affinity is often considered too strong and cannot be significantly modulated without losing activity on the enzymatic target, farnesyl pyrophosphate synthase (FPPS). Furthermore, bisphosphonate bone affinity comes at the expense of very low and variable oral bioavailability. FPPS inhibitors were developed with a monophosphonate as a bone-affinity tag that confers moderate affinity to bone, which can furthermore be tuned to the desired level, and the relationship between structure and bone affinity was evaluated by using an NMR-based bone-binding assay. The concept of targeting drugs to bone with moderate affinity, while retaining oral bioavailability, has broad application to a variety of other bone-targeted drugs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface acoustic wave coding for orthogonal frequency coded devices

NASA Technical Reports Server (NTRS)

Malocha, Donald (Inventor); Kozlovski, Nikolai (Inventor)

2011-01-01

Methods and systems for coding SAW OFC devices to mitigate code collisions in a wireless multi-tag system. Each device producing plural stepped frequencies as an OFC signal with a chip offset delay to increase code diversity. A method for assigning a different OCF to each device includes using a matrix based on the number of OFCs needed and the number chips per code, populating each matrix cell with OFC chip, and assigning the codes from the matrix to the devices. The asynchronous passive multi-tag system includes plural surface acoustic wave devices each producing a different OFC signal having the same number of chips and including a chip offset time delay, an algorithm for assigning OFCs to each device, and a transceiver to transmit an interrogation signal and receive OFC signals in response with minimal code collisions during transmission.

Quantitative protein expression analysis of CLL B cells from mutated and unmutated IgV(H) subgroups using acid-cleavable isotope-coded affinity tag reagents.

PubMed

Barnidge, David R; Jelinek, Diane F; Muddiman, David C; Kay, Neil E

2005-01-01

Relative protein expression levels were compared in leukemic B cells from two patients with chronic lymphocytic leukemia (CLL) having either mutated (M-CLL) or unmutated (UM-CLL) immunoglobulin variable heavy chain genes (IgV(H)). Cells were separated into cytosol and membrane protein fractions then labeled with acid-cleavable ICAT reagents (cICAT). Labeled proteins were digested with trypsin then subjected to SCX and affinity chromatography followed by LC-ESI-MS/MS analysis on a linear ion trap mass spectrometer. A total of 9 proteins from the cytosol fraction and 4 from the membrane fraction showed a 3-fold or greater difference between M-CLL and UM-CLL and a subset of these were examined by Western blot where results concurred with cICAT abundance ratios. The abundance of one of the proteins in particular, the mitochondrial membrane protein cytochrome c oxidase subunit COX G was examined in 6 M-CLL and 6 UM-CLL patients using western blot and results showed significantly greater levels (P < 0.001) in M-CLL patients vs UM-CLL patients. These results demonstrate that stable isotope labeling and mass spectrometry can complement 2D gel electrophoresis and gene microarray technologies for identifying putative and perhaps unique prognostic markers in CLL.

Neutron-Encoded Protein Quantification by Peptide Carbamylation

NASA Astrophysics Data System (ADS)

Ulbrich, Arne; Merrill, Anna E.; Hebert, Alexander S.; Westphall, Michael S.; Keller, Mark P.; Attie, Alan D.; Coon, Joshua J.

2014-01-01

We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.

Investigation of the Mechanism of Electron Capture and Electron Transfer Dissociation of Peptides with a Covalently Attached Free Radical Hydrogen Atom Scavenger.

PubMed

Sohn, Chang Ho; Yin, Sheng; Peng, Ivory; Loo, Joseph A; Beauchamp, J L

2015-11-15

The mechanisms of electron capture and electron transfer dissociation (ECD and ETD) are investigated by covalently attaching a free-radical hydrogen atom scavenger to a peptide. The 2,2,6,6-tetramethylpiperidin-l-oxyl (TEMPO) radical was chosen as the scavenger due to its high hydrogen atom affinity (ca. 280 kJ/mol) and low electron affinity (ca. 0.45 ev), and was derivatized to the model peptide, FQX TEMPO EEQQQTEDELQDK. The X TEMPO residue represents a cysteinyl residue derivatized with an acetamido-TEMPO group. The acetamide group without TEMPO was also examined as a control. The gas phase proton affinity (882 kJ/mol) of TEMPO is similar to backbone amide carbonyls (889 kJ/mol), minimizing perturbation to internal solvation and sites of protonation of the derivatized peptides. Collision induced dissociation (CID) of the TEMPO tagged peptide dication generated stable odd-electron b and y type ions without indication of any TEMPO radical induced fragmentation initiated by hydrogen abstraction. The type and abundance of fragment ions observed in the CID spectra of the TEMPO and acetamide tagged peptides are very similar. However, ECD of the TEMPO labeled peptide dication yielded no backbone cleavage. We propose that a labile hydrogen atom in the charge reduced radical ions is scavenged by the TEMPO radical moiety, resulting in inhibition of N-C α backbone cleavage processes. Supplemental activation after electron attachment (ETcaD) and CID of the charge-reduced precursor ion generated by electron transfer of the TEMPO tagged peptide dication produced a series of b + H (b H ) and y + H (y H ) ions along with some c ions having suppressed intensities, consistent with stable O-H bond formation at the TEMPO group. In summary, the results indicate that ECD and ETD backbone cleavage processes are inhibited by scavenging of a labile hydrogen atom by the localized TEMPO radical moiety. This observation supports the conjecture that ECD and ETD processes involve long-lived intermediates formed by electron capture/transfer in which a labile hydrogen atom is present and plays a key role with low energy processes leading to c and z ion formation. Ab initio and density functional calculations are performed to support our conclusion, which depends most importantly on the proton affinity, electron affinity and hydrogen atom affinity of the TEMPO moiety.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Design of Distortion-Invariant Optical ID Tags for Remote Identification and Verification of Objects

NASA Astrophysics Data System (ADS)

Pérez-Cabré, Elisabet; Millán, María Sagrario; Javidi, Bahram

Optical identification (ID) tags [1] have a promising future in a number of applications such as the surveillance of vehicles in transportation, control of restricted areas for homeland security, item tracking on conveyor belts or other industrial environment, etc. More specifically, passive optical ID tag [1] was introduced as an optical code containing a signature (that is, a characteristic image or other relevant information of the object), which permits its real-time remote detection and identification. Since their introduction in the literature [1], some contributions have been proposed to increase their usefulness and robustness. To increase security and avoid counterfeiting, the signature was introduced in the optical code as an encrypted function [2-5] following the double-phase encryption technique [6]. Moreover, the design of the optical ID tag was done in such a way that tolerance to variations in scale and rotation was achieved [2-5]. To do that, the encrypted information was multiplexed and distributed in the optical code following an appropriate topology. Further studies were carried out to analyze the influence of different sources of noise. In some proposals [5, 7], the designed ID tag consists of two optical codes where the complex-valued encrypted signature was separately introduced in two real-valued functions according to its magnitude and phase distributions. This solution was introduced to overcome some difficulties in the readout of complex values in outdoors environments. Recently, the fully phase encryption technique [8] has been proposed to increase noise robustness of the authentication system.

In vivo phosphorylation of a peptide tag for protein purification.

PubMed

Goux, Marine; Fateh, Amina; Defontaine, Alain; Cinier, Mathieu; Tellier, Charles

2016-05-01

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag. The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III). The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

NASA Technical Reports Server (NTRS)

Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

2000-01-01

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

A set of ligation-independent in vitro translation vectors for eukaryotic protein production.

PubMed

Bardóczy, Viola; Géczi, Viktória; Sawasaki, Tatsuya; Endo, Yaeta; Mészáros, Tamás

2008-03-27

The last decade has brought the renaissance of protein studies and accelerated the development of high-throughput methods in all aspects of proteomics. Presently, most protein synthesis systems exploit the capacity of living cells to translate proteins, but their application is limited by several factors. A more flexible alternative protein production method is the cell-free in vitro protein translation. Currently available in vitro translation systems are suitable for high-throughput robotic protein production, fulfilling the requirements of proteomics studies. Wheat germ extract based in vitro translation system is likely the most promising method, since numerous eukaryotic proteins can be cost-efficiently synthesized in their native folded form. Although currently available vectors for wheat embryo in vitro translation systems ensure high productivity, they do not meet the requirements of state-of-the-art proteomics. Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags. We designed four ligation independent cloning (LIC) vectors for wheat germ extract based in vitro protein translation. In these constructs, the RNA transcription is driven by T7 or SP6 phage polymerase and two TEV protease cleavable affinity tags can be added to aid protein purification. To evaluate our improved vectors, a plant mitogen activated protein kinase was cloned in all four constructs. Purification of this eukaryotic protein kinase demonstrated that all constructs functioned as intended: insertion of PCR fragment by LIC worked efficiently, affinity purification of translated proteins by GST-Sepharose or MagneHis particles resulted in high purity kinase, and the affinity tags could efficiently be removed under different reaction conditions. Furthermore, high in vitro kinase activity testified of proper folding of the purified protein. Four newly designed in vitro translation vectors have been constructed which allow fast and parallel cloning and protein purification, thus representing useful molecular tools for high-throughput production of eukaryotic proteins.

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2017-01-01

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

PubMed

Chen, Gen-Hung; Yin, Li-Jung; Chiang, I-Hua; Jiang, Shann-Tzong

2008-12-01

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

Generic tags for Mn(ii) and Gd(iii) spin labels for distance measurements in proteins.

PubMed

Yang, Yin; Gong, Yan-Jun; Litvinov, Aleksei; Liu, Hong-Kai; Yang, Feng; Su, Xun-Cheng; Goldfarb, Daniella

2017-10-11

High-affinity chelating tags for Gd(iii) and Mn(ii) ions that provide valuable high-resolution distance restraints for biomolecules were used as spin labels for double electron-electron resonance (DEER) measurements. The availability of a generic tag that can bind both metal ions and provide a narrow and predictable distance distribution for both ions is attractive owing to their different EPR-related characteristics. Herein we introduced two paramagnetic tags, 4PSPyMTA and 4PSPyNPDA, which are conjugated to cysteine residues through a stable thioether bond, forming a short and, depending on the metal ion coordination mode, a rigid tether with the protein. These tags exhibit high affinity for both Mn(ii) and Gd(iii) ions. The DEER performance of the 4PSPyMTA and 4PSPyNPDA tags, in complex with Gd(iii) or Mn(ii), was evaluated for three double cysteine mutants of ubiquitin, and the Gd(iii)-Gd(iii) and Mn(ii)-Mn(ii) distance distributions they generated were compared. All three Gd(iii) complexes of the ubiquitin-PyMTA and ubiquitin-PyNPDA conjugates produced similar and expected distance distributions. In contrast, significant variations in the maxima and widths of the distance distributions were observed for the Mn(ii) analogs. Furthermore, whereas PyNPDA-Gd(iii) and PyNPDA-Mn(ii) delivered similar distance distributions, appreciable differences were observed for two mutants with PyMTA, with the Mn(ii) analog exhibiting a broader distance distribution and shorter distances. ELDOR (electron-electron double resonance)-detected NMR measurements revealed some distribution in the Mn(ii) coordination environment for the protein conjugates of both tags but not for the free tags. The broader distance distributions generated by 4PSPyMTA-Mn(ii), as compared with Gd(iii), were attributed to the distributed location of the Mn(ii) ion within the PyMTA chelate owing to its smaller size and lower coordination number that leave the pyridine nitrogen uncoordinated. Accordingly, in terms of distance resolution, 4PSPyNPDA can serve as an effective generic tag for Gd(iii) and Mn(ii), whereas 4PSPyMTA is efficient for Gd(iii) only. This comparison between Gd(iii) and Mn(ii) suggests that PyMTA model compounds may not predict sufficiently well the performance of PyMTA-Mn(ii) as a tag for high-resolution distance measurements in proteins because the protein environment can influence its coordination mode.

A Synthesis of Tagging Studies Examining the Behaviour and Survival of Anadromous Salmonids in Marine Environments

PubMed Central

Drenner, S. Matthew; Clark, Timothy D.; Whitney, Charlotte K.; Martins, Eduardo G.; Cooke, Steven J.; Hinch, Scott G.

2012-01-01

This paper synthesizes tagging studies to highlight the current state of knowledge concerning the behaviour and survival of anadromous salmonids in the marine environment. Scientific literature was reviewed to quantify the number and type of studies that have investigated behaviour and survival of anadromous forms of Pacific salmon (Oncorhynchus spp.), Atlantic salmon (Salmo salar), brown trout (Salmo trutta), steelhead (Oncorhynchus mykiss), and cutthroat trout (Oncorhynchus clarkii). We examined three categories of tags including electronic (e.g. acoustic, radio, archival), passive (e.g. external marks, Carlin, coded wire, passive integrated transponder [PIT]), and biological (e.g. otolith, genetic, scale, parasites). Based on 207 papers, survival rates and behaviour in marine environments were found to be extremely variable spatially and temporally, with some of the most influential factors being temperature, population, physiological state, and fish size. Salmonids at all life stages were consistently found to swim at an average speed of approximately one body length per second, which likely corresponds with the speed at which transport costs are minimal. We found that there is relatively little research conducted on open-ocean migrating salmonids, and some species (e.g. masu [O. masou] and amago [O. rhodurus]) are underrepresented in the literature. The most common forms of tagging used across life stages were various forms of external tags, coded wire tags, and acoustic tags, however, the majority of studies did not measure tagging/handling effects on the fish, tag loss/failure, or tag detection probabilities when estimating survival. Through the interdisciplinary application of existing and novel technologies, future research examining the behaviour and survival of anadromous salmonids could incorporate important drivers such as oceanography, tagging/handling effects, predation, and physiology. PMID:22431962

A synthesis of tagging studies examining the behaviour and survival of anadromous salmonids in marine environments.

PubMed

Drenner, S Matthew; Clark, Timothy D; Whitney, Charlotte K; Martins, Eduardo G; Cooke, Steven J; Hinch, Scott G

2012-01-01

This paper synthesizes tagging studies to highlight the current state of knowledge concerning the behaviour and survival of anadromous salmonids in the marine environment. Scientific literature was reviewed to quantify the number and type of studies that have investigated behaviour and survival of anadromous forms of Pacific salmon (Oncorhynchus spp.), Atlantic salmon (Salmo salar), brown trout (Salmo trutta), steelhead (Oncorhynchus mykiss), and cutthroat trout (Oncorhynchus clarkii). We examined three categories of tags including electronic (e.g. acoustic, radio, archival), passive (e.g. external marks, Carlin, coded wire, passive integrated transponder [PIT]), and biological (e.g. otolith, genetic, scale, parasites). Based on 207 papers, survival rates and behaviour in marine environments were found to be extremely variable spatially and temporally, with some of the most influential factors being temperature, population, physiological state, and fish size. Salmonids at all life stages were consistently found to swim at an average speed of approximately one body length per second, which likely corresponds with the speed at which transport costs are minimal. We found that there is relatively little research conducted on open-ocean migrating salmonids, and some species (e.g. masu [O. masou] and amago [O. rhodurus]) are underrepresented in the literature. The most common forms of tagging used across life stages were various forms of external tags, coded wire tags, and acoustic tags, however, the majority of studies did not measure tagging/handling effects on the fish, tag loss/failure, or tag detection probabilities when estimating survival. Through the interdisciplinary application of existing and novel technologies, future research examining the behaviour and survival of anadromous salmonids could incorporate important drivers such as oceanography, tagging/handling effects, predation, and physiology.

Cobalt carbonyl complexes as probes for alkyne-tagged lipids[S

PubMed Central

Tallman, Keri A.; Armstrong, Michelle D.; Milne, Stephen B.; Marnett, Lawrence J.; Brown, H. Alex; Porter, Ned A.

2013-01-01

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection. PMID:23307946

Stability of the neurotensin receptor NTS1 free in detergent solution and immobilized to affinity resin.

PubMed

White, Jim F; Grisshammer, Reinhard

2010-09-07

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.

Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

PubMed Central

Menger, Katja E.; James, Andrew M.; Cochemé, Helena M.; Harbour, Michael E.; Chouchani, Edward T.; Ding, Shujing; Fearnley, Ian M.; Partridge, Linda; Murphy, Michael P.

2015-01-01

Summary Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster. PMID:26095360

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

PubMed

Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

2015-09-01

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Soluble expression and one-step purification of recombinant mouse interferon-λ3 in Escherichia coli.

PubMed

Wang, Y Q; Zhou, M; Zeng, L M; Gao, Q Y; Yuan, X L; Li, Y; Li, M C

2015-02-01

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

Tracking the potyviral P1 protein in Nicotiana benthamiana plants during plum pox virus infection.

PubMed

Vozárová, Z; Glasa, M; Šubr, Z W

The P1 protein is derived from the N terminus of potyvirus-coded polyprotein. In addition to the proteolytic activity essential for its maturation, it probably participates in suppression of host defense and/or in virus replication. Clear validation of the P1 in vivo function(s), however, is not yet available. We applied an infectious cDNA clone of plum pox virus (PPV), where the P1 was N-fused with a hexahistidine tag, to trace this protein in Nicotiana benthamiana plants during the PPV infection. Immunoblot analysis with the anti-his antibody showed a diffuse band corresponding to the molecular weight about 70-80 kDa (about twice larger than expected) in the root samples from early stage of infection. This signal culminated on the sixth day post inoculation, later it rapidly disappeared. Sample denaturation by boiling in SDS before centrifugal clarification was essential, indicating strong affinity of P1-his to some plant compound sedimenting with the tissue and cell debris.

Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules.

PubMed

Han, M; Gao, X; Su, J Z; Nie, S

2001-07-01

Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots (zinc sulfide-capped cadmium selenide nanocrystals) into polymeric microbeads at precisely controlled ratios. Their novel optical properties (e.g., size-tunable emission and simultaneous excitation) render these highly luminescent quantum dots (QDs) ideal fluorophores for wavelength-and-intensity multiplexing. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic measurements indicate that the QD-tagged beads are highly uniform and reproducible, yielding bead identification accuracies as high as 99.99% under favorable conditions. DNA hybridization studies demonstrate that the coding and target signals can be simultaneously read at the single-bead level. This spectral coding technology is expected to open new opportunities in gene expression studies, high-throughput screening, and medical diagnostics.

Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

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

Zhang, Xinxin; Zhang, Min; Takano, Tetsuo

Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membranemore » and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.« less

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

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

PubMed

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

2017-01-01

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

Affinity monolith-integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis.

PubMed

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T

2008-07-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Affinity Monolith-Integrated Poly(methyl Methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

PubMed Central

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T.

2008-01-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis. PMID:18479142

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

PubMed

Trachman, Robert J; Abdolahzadeh, Amir; Andreoni, Alessio; Cojocaru, Razvan; Knutson, Jay R; Ryckelynck, Michael; Unrau, Peter J; Ferré-D'Amaré, Adrian R

2018-05-24

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

NASA Astrophysics Data System (ADS)

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-08-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

Preparation of next-generation sequencing libraries using Nextera™ technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition.

PubMed

Caruccio, Nicholas

2011-01-01

DNA library preparation is a common entry point and bottleneck for next-generation sequencing. Current methods generally consist of distinct steps that often involve significant sample loss and hands-on time: DNA fragmentation, end-polishing, and adaptor-ligation. In vitro transposition with Nextera™ Transposomes simultaneously fragments and covalently tags the target DNA, thereby combining these three distinct steps into a single reaction. Platform-specific sequencing adaptors can be added, and the sample can be enriched and bar-coded using limited-cycle PCR to prepare di-tagged DNA fragment libraries. Nextera technology offers a streamlined, efficient, and high-throughput method for generating bar-coded libraries compatible with multiple next-generation sequencing platforms.

System and method for simultaneously collecting serial number information from numerous identity tags

DOEpatents

Doty, Michael A.

1997-01-01

A system and method for simultaneously collecting serial number information reports from numerous colliding coded-radio-frequency identity tags. Each tag has a unique multi-digit serial number that is stored in non-volatile RAM. A reader transmits an ASCII coded "D" character on a carrier of about 900 MHz and a power illumination field having a frequency of about 1.6 Ghz. A one MHz tone is modulated on the 1.6 Ghz carrier as a timing clock for a microprocessor in each of the identity tags. Over a thousand such tags may be in the vicinity and each is powered-up and clocked by the 1.6 Ghz power illumination field. Each identity tag looks for the "D" interrogator modulated on the 900 MHz carrier, and each uses a digit of its serial number to time a response. Clear responses received by the reader are repeated for verification. If no verification or a wrong number is received by any identity tag, it uses a second digital together with the first to time out a more extended period for response. Ultimately, the entire serial number will be used in the worst case collision environments; and since the serial numbers are defined as being unique, the final possibility will be successful because a clear time-slot channel will be available.

System and method for simultaneously collecting serial number information from numerous identity tags

DOEpatents

Doty, M.A.

1997-01-07

A system and method are disclosed for simultaneously collecting serial number information reports from numerous colliding coded-radio-frequency identity tags. Each tag has a unique multi-digit serial number that is stored in non-volatile RAM. A reader transmits an ASCII coded ``D`` character on a carrier of about 900 MHz and a power illumination field having a frequency of about 1.6 Ghz. A one MHz tone is modulated on the 1.6 Ghz carrier as a timing clock for a microprocessor in each of the identity tags. Over a thousand such tags may be in the vicinity and each is powered-up and clocked by the 1.6 Ghz power illumination field. Each identity tag looks for the ``D`` interrogator modulated on the 900 MHz carrier, and each uses a digit of its serial number to time a response. Clear responses received by the reader are repeated for verification. If no verification or a wrong number is received by any identity tag, it uses a second digital together with the first to time out a more extended period for response. Ultimately, the entire serial number will be used in the worst case collision environments; and since the serial numbers are defined as being unique, the final possibility will be successful because a clear time-slot channel will be available. 5 figs.

Sequence-Specific Affinity Chromatography of Bacterial Small Regulatory RNA-Binding Proteins from Bacterial Cells.

PubMed

Gans, Jonathan; Osborne, Jonathan; Cheng, Juliet; Djapgne, Louise; Oglesby-Sherrouse, Amanda G

2018-01-01

Bacterial small RNA molecules (sRNAs) are increasingly recognized as central regulators of bacterial stress responses and pathogenesis. In many cases, RNA-binding proteins are critical for the stability and function of sRNAs. Previous studies have adopted strategies to genetically tag an sRNA of interest, allowing isolation of RNA-protein complexes from cells. Here we present a sequence-specific affinity purification protocol that requires no prior genetic manipulation of bacterial cells, allowing isolation of RNA-binding proteins bound to native RNA molecules.

ONE-STEP METAL-AFFINITY PURIFICATION OF HISTIDINE-TAGGED PROTEINS BY TEMPERATURE-TRIGGERED PRECIPITATION. (R829606)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

PubMed

Tong, Jiefei; Cao, Biyin; Martyn, Gregory D; Krieger, Jonathan R; Taylor, Paul; Yates, Bradley; Sidhu, Sachdev S; Li, Shawn S C; Mao, Xinliang; Moran, Michael F

2017-03-01

Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ocean Heat Content Reveals Secrets of Fish Migrations

PubMed Central

Luo, Jiangang; Ault, Jerald S.; Shay, Lynn K.; Hoolihan, John P.; Prince, Eric D.; Brown, Craig A.; Rooker, Jay R.

2015-01-01

For centuries, the mechanisms surrounding spatially complex animal migrations have intrigued scientists and the public. We present a new methodology using ocean heat content (OHC), a habitat metric that is normally a fundamental part of hurricane intensity forecasting, to estimate movements and migration of satellite-tagged marine fishes. Previous satellite-tagging research of fishes using archival depth, temperature and light data for geolocations have been too coarse to resolve detailed ocean habitat utilization. We combined tag data with OHC estimated from ocean circulation and transport models in an optimization framework that substantially improved geolocation accuracy over SST-based tracks. The OHC-based movement track provided the first quantitative evidence that many of the tagged highly migratory fishes displayed affinities for ocean fronts and eddies. The OHC method provides a new quantitative tool for studying dynamic use of ocean habitats, migration processes and responses to environmental changes by fishes, and further, improves ocean animal tracking and extends satellite-based animal tracking data for other potential physical, ecological, and fisheries applications. PMID:26484541

Designed Proteins as Novel Imaging Reagents in Living Escherichia coli.

PubMed

Pratt, Susan E; Speltz, Elizabeth B; Mochrie, Simon G J; Regan, Lynne

2016-09-02

Fluorescence imaging is a powerful tool to study protein function in living cells. Here, we introduce a novel imaging strategy that is fully genetically encodable, does not require the use of exogenous substrates, and adds a minimally disruptive tag to the protein of interest (POI). Our method was based on a set of designed tetratricopeptide repeat affinity proteins (TRAPs) that specifically and reversibly interact with a short, extended peptide tag. We co-expressed the TRAPs fused to fluorescent proteins (FPs) and the peptide tags fused to the POIs. We illustrated the method using the Escherichia coli protein FtsZ and showed that our system could track distinct FtsZ structures under both low and high expression conditions in live cells. We anticipate that our imaging strategy will be a useful tool for imaging the subcellular localization of many proteins, especially those recalcitrant to imaging by direct tagging with FPs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.

PubMed

Chen, Ying-Hui; Wang, Gao-Yuan; Hao, Hao-Chao; Chao, Chun-Jiang; Wang, Yamei; Jin, Quan-Wen

2017-03-01

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe , we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale. © 2017. Published by The Company of Biologists Ltd.

Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5.

PubMed

Singh, Ram Sarup; Yadav, Mukesh

2012-07-01

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b⁺ expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5 mM) induction for 5 h at 37 °C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni-NTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH 8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35 °C. The enzyme showed apparent K(m) and V(max) values for L-aspartate as 2.01 mM and 114 U/mg, respectively. The enzyme was stable in pH range of 6.5-9.5 and temperature up to 45 °C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51 kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.

Integrity of N- and C-termini is important for E. coli Hsp31 chaperone activity

PubMed Central

Sastry, M S R; Zhou, Weibin; Baneyx, François

2009-01-01

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging. PMID:19517531

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Swarmathon 2017

NASA Image and Video Library

2017-04-19

A Swarmie robot finds a "resource" cube marked with an AprilTag, similar to a barcode. In the Swarmathon competition at the Kennedy Space Center Visitor Complex, students were asked to develop computer code for the small robots, programming them to look for "resources" in the form of cubes with AprilTags.

Analysis of the STAT3 interactome using in-situ biotinylation and SILAC.

PubMed

Blumert, Conny; Kalkhof, Stefan; Brocke-Heidrich, Katja; Kohajda, Tibor; von Bergen, Martin; Horn, Friedemann

2013-12-06

Signal transducer and activator of transcription 3 (STAT3) is activated by a variety of cytokines and growth factors. To generate a comprehensive data set of proteins interacting specifically with STAT3, we applied stable isotope labeling with amino acids in cell culture (SILAC). For high-affinity pull-down using streptavidin, we fused STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag), which did not affect STAT3 functions. By this approach, 3642 coprecipitated proteins were detected in human embryonic kidney-293 cells. Filtering using statistical and functional criteria finally extracted 136 proteins as putative interaction partners of STAT3. Both, a physical interaction network analysis and the enrichment of known and predicted interaction partners suggested that our filtering criteria successfully enriched true STAT3 interactors. Our approach identified numerous novel interactors, including ones previously predicted to associate with STAT3. By reciprocal coprecipitation, we were able to verify the physical association between STAT3 and selected interactors, including the novel interaction with TOX4, a member of the TOX high mobility group box family. Applying the same method, we next investigated the activation-dependency of the STAT3 interactome. Again, we identified both known and novel interactions. Thus, our approach allows to study protein-protein interaction effectively and comprehensively. The location, activity, function, degradation, and synthesis of proteins are significantly regulated by interactions of proteins with other proteins, biopolymers and small molecules. Thus, the comprehensive characterization of interactions of proteins in a given proteome is the next milestone on the path to understanding the biochemistry of the cell. In order to generate a comprehensive interactome dataset of proteins specifically interacting with a selected bait protein, we fused our bait protein STAT3 with a short peptide tag allowing biotinylation in situ (bio-tag). This bio-tag allows an affinity pull-down using streptavidin but affected neither the activation of STAT3 by tyrosine phosphorylation nor its transactivating potential. We combined SILAC for accurate relative protein quantification, subcellular fractionation to increase the coverage of interacting proteins, high-affinity pull-down and a stringent filtering method to successfully analyze the interactome of STAT3. With our approach we confirmed several already known and identified numerous novel STAT3 interactors. The approach applied provides a rapid and effective method, which is broadly applicable for studying protein-protein interactions and their dependency on post-translational modifications. © 2013. Published by Elsevier B.V. All rights reserved.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Expression, purification, and functional analysis of the C-terminal domain of Herbaspirillum seropedicae NifA protein.

PubMed

Monteiro, Rose A; Souza, Emanuel M; Geoffrey Yates, M; Steffens, M Berenice R; Pedrosa, Fábio O; Chubatsu, Leda S

2003-02-01

The Herbaspirillum seropedicae NifA protein is responsible for nif gene expression. The C-terminal domain of the H. seropedicae NifA protein, fused to a His-Tag sequence (His-Tag-C-terminal), was over-expressed and purified by metal-affinity chromatography to yield a highly purified and active protein. Band-shift assays showed that the NifA His-Tag-C-terminal bound specifically to the H. seropedicae nifB promoter region in vitro. In vivo analysis showed that this protein inhibited the Central + C-terminal domains of NifA protein from activating the nifH promoter of K. pneumoniae in Escherichia coli, indicating that the protein must be bound to the NifA-binding site (UAS site) at the nifH promoter region to activate transcription. Copyright 2002 Elsevier Science (USA)

Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease.

PubMed

Li, Junhua; Zhang, Yang; Shen, Fei; Yang, Yanjun

2012-10-15

A fusion tag that can be purified by the cheap ion-exchanger based on the ionic binding force may provide a cost-effective scheme over other affinity fusion tags. Small ubiquitin-like modifier (SUMO) protease derived from Saccharomyces cerevisiae was fused with a poly lysine tag containing 10 lysine residues at its C-terminus and then expressed in Escherichia coli. The ionic binding force provided by the ploy lysine tag allowed the selective recovery of the small ubiquitin-like modifier protease from recombinant E. coli cell extracts. A preliminary comparative study of the adsorption and elution of poly lysine tagged SUMO protease on Amberlite Cobalamion and magnetite carboxymethyl chitosan nanoparticles was performed. Amberlite Cobalamion and magnetite nanoparticles had the similar elution profile due to the common functional groups - carboxyl groups. The maximum dynamic adsorption capacity of Amberlite Cobalamion and magnetite nanoparticles reached 36.8 and 211.4 mg/g, respectively. The lysine-tagged protease can be simply purified by magnetite nanoparticles from cell extracts with higher purity than that by Amberlite Cobalamion. The superparamagnetic nanoparticles possess the advantages of highly specific, fast and excellent binding of a larger amount of lysine tagged SUMO modifier protease, and it is also easier to separate from the crude biological process liquors compared with the conventional separation techniques of polycationic amino acids fusion proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

A new pH-responsive peptide tag for protein purification.

PubMed

Nonaka, Takahiro; Tsurui, Noriko; Mannen, Teruhisa; Kikuchi, Yoshimi; Shiraki, Kentaro

2018-06-01

This paper describes a new pH-responsive peptide tag that adds a protein reversible precipitation and redissolution character. This peptide tag is a part of a cell surface protein B (CspB) derived from Corynebacterium glutamicum. Proinsulin that genetically fused with a peptide of N-terminal 6, 17, 50, or 250 amino acid residues of CspB showed that the reversible precipitation and redissolution depended on the pH. The transition occurred within a physiological and narrow pH range. A CspB50 tag comprising 50 amino acid residues of N-terminal CspB was further evaluated as a representative using other pharmaceutical proteins. Below pH 6.8, almost all CspB50-Teriparatide fusion formed an aggregated state. Subsequent addition of alkali turned the cloudy protein solution transparent above pH 7.3, in which almost all the CspB50-Teriparatide fusion redissolved. The CspB50-Bivalirudin fusion showed a similar behavior with slightly different pH range. This tag is offering a new protein purification method based on liquid-solid separation which does not require an affinity ligand. This sharp response around neutral pH is useful as a pH-responsive tag for the purification of unstable proteins at a non-physiological pH. Copyright © 2018 Elsevier Inc. All rights reserved.

Supporting Situated Learning Based on QR Codes with Etiquetar App: A Pilot Study

ERIC Educational Resources Information Center

Camacho, Miguel Olmedo; Pérez-Sanagustín, Mar; Alario-Hoyos, Carlos; Soldani, Xavier; Kloos, Carlos Delgado; Sayago, Sergio

2014-01-01

EtiquetAR is an authoring tool for supporting the design and enactment of situated learning experiences based on QR tags. Practitioners use etiquetAR for creating, managing and personalizing collections of QR codes with special properties: (1) codes can have more than one link pointing at different multimedia resources, (2) codes can be updated…

Addition of six-His-tagged peptide to the C terminus of adeno-associated virus VP3 does not affect viral tropism or production.

PubMed

Zhang, Huang-Ge; Xie, Jinfu; Dmitriev, Igor; Kashentseva, Elena; Curiel, David T; Hsu, Hui-Chen; Mountz, John D

2002-12-01

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective. To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6xHis tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6xHis)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6xHis-tagged VP3mutant recombinant AAV. The 6xHis-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6xHis-modified AAV were equivalent to those of wild-type particles. The 6xHis-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6xHis tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6xHis-tagged AAV VP3 capsid protein and to utilize the 6xHis-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

PubMed

Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

2015-02-01

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of selected binding domains for the analysis of ubiquitinated proteomes

PubMed Central

Nakayasu, Ernesto S.; Ansong, Charles; Brown, Joseph N.; Yang, Feng; Lopez-Ferrer, Daniel; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.

2013-01-01

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising, but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ∼200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle. PMID:23649778

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

PubMed

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Electronic tagging and integrated product intelligence

NASA Astrophysics Data System (ADS)

Swerdlow, Martin; Weeks, Brian

1996-03-01

The advent of 'intelligent,' electronic data bearing tags is set to revolutionize the way industrial and retail products are identified and tracked throughout their life cycles. The dominant system for unique identification today is the bar code, which is based on printed symbology and regulated by the International Article Numbering Association. Bar codes provide users with significant operational advantages and generate considerable added value to packaging companies, product manufacturers, distributors and retailers, across supply chains in many different sectors, from retailing, to baggage handling and industrial components, e.g., for vehicles or aircraft. Electronic tags offer the potential to: (1) record and store more complex data about the product or any modifications which occur during its life cycle; (2) access (and up-date) stored data in real time in a way which does not involve contact with the product or article; (3) overcome the limitations imposed by systems which rely on line-of-sight access to stored data. Companies are now beginning to consider how electronic data tags can be used, not only to improve the efficiency of their supply chain processes, but also to revolutionize the way they do business. This paper reviews the applications and business opportunities for electronic tags and outlines CEST's strategy for achieving an 'open' standard which will ensure that tags from different vendors can co-exist on an international basis.

The chemical basis for the origin of the genetic code and the process of protein synthesis

NASA Technical Reports Server (NTRS)

1982-01-01

The major thrust is to understand just how the process of protein synthesis, including that very important aspect, genetic coding, came to be. Two aspects of the problem: the chemistry of active aminoacyl species; and affinities between amino acids and nucleotides, and specifically, how these affinities might affect the chemistry between the two are stressed.

HaloTag technology for specific and covalent labeling of fusion proteins.

PubMed

Benink, Hélène A; Urh, Marjeta

2015-01-01

Appending proteins of interest to fluorescent protein tags such as GFP has revolutionized how proteins are studied in the cellular environment. Over the last few decades many varieties of fluorescent proteins have been generated, each bringing new capability to research. However, taking full advantage of standard fluorescent proteins with advanced and differential features requires significant effort on the part of the researcher. This approach necessitates that many genetic fusions be generated and confirmed to function properly in cells with the same protein of interest. To lessen this burden, a newer category of protein fusion tags termed "self-labeling protein tags" has been developed. This approach utilizes a single protein tag, the function of which can be altered by attaching various chemical moieties (fluorescent labels, affinity handles, etc.). In this way a single genetically encoded protein fusion can easily be given functional diversity and adaptability as supplied by synthetic chemistry. Here we present protein labeling methods using HaloTag technology; comprised of HaloTag protein and the collection of small molecules designed to bind it specifically and provide it with varied functionalities. For imaging purposes these small molecules, termed HaloTag ligands, contain distinct fluorophores. Due to covalent and rapid binding between HaloTag protein and its ligands, labeling is permanent and efficient. Many of these ligands have been optimized for permeability across cellular membranes allowing for live cell labeling and imaging analysis. Nonpermeable ligands have also been developed for specific labeling of surface proteins. Overall, HaloTag is a versatile technology that empowers the end user to label a protein of interest with the choice of different fluorophores while alleviating the need for generation of multiple genetic fusions.

A two-step strategy to visually identify molecularly imprinted polymers for tagged proteins.

PubMed

Brandis, Alexander; Partouche, Eran; Yechezkel, Tamar; Salitra, Yoseph; Shkoulev, Vladimir; Scherz, Avigdor; Grynszpan, Flavio

2017-08-01

A practical and relatively simple method to identify molecularly imprinted polymers capable of binding proteins via the molecular tagging (epitope-like) approach has been developed. In our two-step method, we first challenge a previously obtained anti-tag molecularly imprinted polymer with a small molecule including the said tag of choice (a biotin derivative as shown here or other) connected to a linker bound to a second biotin moiety. An avidin molecule partially decorated with fluorescent labels is then allowed to bind the available biotin derivative associated with the polymer matrix. At the end of this simple process, and after washing off all the low-affinity binding molecules from the polymer matrix, only suitable molecularly imprinted polymers binding avidin through its previously acquired small molecule tag (or epitope-like probe, in a general case) will remain fluorescent. For confirmation, we tested the selective performance of the anti-biotin molecularly imprinted polymer binding it to biotinylated alkaline phosphatase. Residual chemical activity of the enzyme on the molecularly imprinted polymer solid support was observed. In all cases, the corresponding nonimprinted polymer controls were inactive. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of Fe3O4@nickel-silicate core-shell nanoparticles for His-tagged enzyme immobilizing agents

NASA Astrophysics Data System (ADS)

Shin, Moo-Kwang; Kang, Byunghoon; Yoon, Nam-Kyung; Kim, Myeong-Hoon; Ki, Jisun; Han, Seungmin; Ahn, Jung-Oh; Haam, Seungjoo

2016-12-01

Immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. Specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various pH and temperatures. Herein, we fabricated Fe3O4 encapsulated ‘sea-urchin’ shaped nickel-silicate nanoparticles with a facile synthetic route. The enzymes were then rapidly and easily immobilized with poly-histidine tags (His-tags) and nickel ion affinity. Porous nickel silicate covered nanoparticles achieved a high immobilization capacity (85 μg mg-1) of His-tagged tobacco etch virus (TEV) protease. To investigate immobilized TEV protease enzymatic activity, we analyzed the cleaved quantity of maltose binding protein-exendin-fused immunoglobulin fusion protein, which connected with the TEV protease-specific cleavage peptide sequence. Moreover, TEV protease immobilized nanocomplexes conveniently removed and recollected from the reactant by applying an external magnetic field, maintained their enzymatic activity after reuse. Therefore, our newly developed nanoplatform for His-tagged enzyme immobilization provides advantageous features for biotechnological industries including recombinant protein processing.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

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

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

Systematic Localization and Identification of SUMOylation Substrates in Knock-In Mice Expressing Affinity-Tagged SUMO1.

PubMed

Tirard, Marilyn; Brose, Nils

2016-01-01

Protein SUMOylation is a posttranslational protein modification that is emerging as a key regulatory process in neurobiology. To date, however, SUMOylation in vivo has only been studied cursorily. Knock-in mice expressing His6-HA-SUMO1 from the Sumo1 locus allow for the highly specific localization and identification of endogenous SUMO1 substrates under physiological and pathophysiological conditions. By making use of the HA-tag and using wild-type mice for highly stringent negative control samples, SUMO1 targets can be specifically localized in and purified from cultured mouse nerve cells and mouse tissues.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

PubMed Central

Tulloch, Lindsay B.; Menzies, Stefanie K.; Fraser, Andrew L.; Gould, Eoin R.; King, Elizabeth F.; Zacharova, Marija K.; Florence, Gordon J.

2017-01-01

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model. PMID:28873407

Feasibility of ultra-wideband SAW RFID tags meeting FCC rules.

PubMed

Härmä, Sanna; Plessky, Victor P; Li, Xianyi; Hartogh, Paul

2009-04-01

We discuss the feasibility of surface acoustic wave (SAW) radio-frequency identification (RFID) tags that rely on ultra-wideband (UWB) technology. We propose a design of a UWB SAW tag, carry out numerical experiments on the device performance, and study signal processing in the system. We also present experimental results for the proposed device and estimate the potentially achievable reading distance. UWB SAW tags will have an extremely small chip size (<0.5 x 1 mm(2)) and a low cost. They also can provide a large number of different codes. The estimated read range for UWB SAW tags is about 2 m with a reader radiating as low as <0.1 mW power levels with an extremely low duty factor.

Phase modulation in RF tag

DOEpatents

Carrender, Curtis Lee; Gilbert, Ronald W.

2007-02-20

A radio frequency (RF) communication system employs phase-modulated backscatter signals for RF communication from an RF tag to an interrogator. The interrogator transmits a continuous wave interrogation signal to the RF tag, which based on an information code stored in a memory, phase-modulates the interrogation signal to produce a backscatter response signal that is transmitted back to the interrogator. A phase modulator structure in the RF tag may include a switch coupled between an antenna and a quarter-wavelength stub; and a driver coupled between the memory and a control terminal of the switch. The driver is structured to produce a modulating signal corresponding to the information code, the modulating signal alternately opening and closing the switch to respectively decrease and increase the transmission path taken by the interrogation signal and thereby modulate the phase of the response signal. Alternatively, the phase modulator may include a diode coupled between the antenna and driver. The modulating signal from the driver modulates the capacitance of the diode, which modulates the phase of the response signal reflected by the diode and antenna.

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

NASA Astrophysics Data System (ADS)

Liu, Fang

Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

Dynamic optical tags

NASA Astrophysics Data System (ADS)

Griggs, Steven P.; Mark, Martin B.; Feldman, Barry J.

2004-07-01

The goal of the DARPA Dynamic Optical Tags (DOTs) program is to develop a small, robust, persistent, 2-way tagging, tracking and locating device that also supports communications at data rates greater than 100 kbps and can be interrogated at significant range. These tags will allow for two-way data exchange and tagging operations in friendly and denied areas. The DOTs will be passive and non-RF. To accomplish this, the DOTs program will develop small, thin, retro-reflecting modulators. The tags will operate for long periods of time (greater than two months) in real-world environmental conditions (-40° to +70° C) and allow for a wide interrogation angle (+/-60°). The tags will be passive (in the sleep mode) for most of the time and only become active when interrogated by a laser with the correct code. Once correctly interrogated, the tags will begin to modulate and retro-reflect the incoming beam. The program will also develop two tag specific transceiver systems that are eye-safe, employ automated scanning algorithms, and are capable of short search and interrogate times.

Aptamer facilitated purification of functional proteins.

PubMed

Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

2018-01-15

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

PubMed

Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

2014-01-16

Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins.

PubMed

Hu, Pingzhao; Janga, Sarath Chandra; Babu, Mohan; Díaz-Mejía, J Javier; Butland, Gareth; Yang, Wenhong; Pogoutse, Oxana; Guo, Xinghua; Phanse, Sadhna; Wong, Peter; Chandran, Shamanta; Christopoulos, Constantine; Nazarians-Armavil, Anaies; Nasseri, Negin Karimi; Musso, Gabriel; Ali, Mehrab; Nazemof, Nazila; Eroukova, Veronika; Golshani, Ashkan; Paccanaro, Alberto; Greenblatt, Jack F; Moreno-Hagelsieb, Gabriel; Emili, Andrew

2009-04-28

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Quantitative proteomic analysis of human breast epithelial cells with differential telomere length

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

Yu, Li-Rong; Chan, King C.; Tahara, Hidetoshi

Telomeres play important functional roles in cell proliferation, cell cycle regulation, and genetic stability, in which telomere length is critical. In this study, quantitative proteome comparisons for the human breast epithelial cells with short and long telomeres (184-hTERT{sub L} vs. 184-hTERT{sub S} and 90P-hTERT{sub L} vs. 90P-hTERT{sub S}), resulting from transfection of the human telomerase reverse transcriptase (hTERT) gene, were performed using cleavable isotope-coded affinity tags. More than 2000 proteins were quantified in each comparative experiment, with approximately 77% of the proteins identified in both analyses. In the cells with long telomeres, significant and consistent alterations were observed in metabolismmore » (amino acid, nucleotide, and lipid metabolism), genetic information transmission (transcription and translation regulation, spliceosome and ribosome complexes), and cell signaling. Interestingly, the DNA excision repair pathway is enhanced, while integrin and its ligands are downregulated in the cells with long telomeres. These results may provide valuable information related to telomere functions.« less

Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Doclet To Synthesize UML

NASA Technical Reports Server (NTRS)

Barry, Matthew R.; Osborne, Richard N.

2005-01-01

The RoseDoclet computer program extends the capability of Java doclet software to automatically synthesize Unified Modeling Language (UML) content from Java language source code. [Doclets are Java-language programs that use the doclet application programming interface (API) to specify the content and format of the output of Javadoc. Javadoc is a program, originally designed to generate API documentation from Java source code, now also useful as an extensible engine for processing Java source code.] RoseDoclet takes advantage of Javadoc comments and tags already in the source code to produce a UML model of that code. RoseDoclet applies the doclet API to create a doclet passed to Javadoc. The Javadoc engine applies the doclet to the source code, emitting the output format specified by the doclet. RoseDoclet emits a Rose model file and populates it with fully documented packages, classes, methods, variables, and class diagrams identified in the source code. The way in which UML models are generated can be controlled by use of new Javadoc comment tags that RoseDoclet provides. The advantage of using RoseDoclet is that Javadoc documentation becomes leveraged for two purposes: documenting the as-built API and keeping the design documentation up to date.

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

DOE PAGES

Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; ...

2015-03-31

Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

Redox regulation of mitochondrial proteins and proteomes by cysteine thiol switches.

PubMed

Nietzel, Thomas; Mostertz, Jörg; Hochgräfe, Falko; Schwarzländer, Markus

2017-03-01

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Reconstitution and functional comparison of purified GlpF and AqpZ, the glycerol and water channels from Escherichia coli.

PubMed

Borgnia, M J; Agre, P

2001-02-27

A large family of membrane channel proteins selective for transport of water (aquaporins) or water plus glycerol (aquaglyceroporins) has been found in diverse life forms. Escherichia coli has two members of this family-a water channel, AqpZ, and a glycerol facilitator, GlpF. Despite having similar primary amino acid sequences and predicted structures, the oligomeric state and solute selectivity of AqpZ and GlpF are disputed. Here we report biochemical and functional characterizations of affinity-purified GlpF and compare it to AqpZ. Histidine-tagged (His-GlpF) and hemagglutinin-tagged (HA-GlpF) polypeptides encoded by a bicistronic construct were expressed in bacteria. HA-GlpF and His-GlpF appear to form oligomers during Ni-nitrilotriacetate affinity purification. Sucrose gradient sedimentation analyses showed that the oligomeric state of octyl glucoside-solubilized GlpF varies: low ionic strength favors subunit dissociation, whereas Mg(2+) stabilizes tetrameric assembly. Reconstitution of affinity-purified GlpF into proteoliposomes increases glycerol permeability more than 100-fold and water permeability up to 10-fold compared with control liposomes. Glycerol and water permeability of GlpF both occur with low Arrhenius activation energies and are reversibly inhibited by HgCl(2). Our studies demonstrate that, unlike AqpZ, a water-selective stable tetramer, purified GlpF exists in multiple oligomeric forms under nondenaturing conditions and is highly permeable to glycerol but less well permeated by water.

Coded wire tag recoveries from pink salmon in Prince William sound salmon fisheries, 1993. Restoration project 93067. Exxon Valdez oil spill restoration project final report

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

Sharr, S.; Peckham, C.J.; Sharp, D.G.

1995-11-01

Coded wire tags applied to pink salmon fry in 1992 at four hatcheries in Prince William Sound were recovered in the commercial catch of 1993 and used to provide inseason estimates of hatchery contributions. These estimates were used by fishery managers to target the numerically superior hatchery returns, and reduce the pressure on oil-damaged wild stocks. Inseason estimates were made in two stages. The postseason analysis revealed that of a catch of 3.51 million pink salmon, 1.12 million were estimated to be of wild origin.

Semantic Annotation of Complex Text Structures in Problem Reports

NASA Technical Reports Server (NTRS)

Malin, Jane T.; Throop, David R.; Fleming, Land D.

2011-01-01

Text analysis is important for effective information retrieval from databases where the critical information is embedded in text fields. Aerospace safety depends on effective retrieval of relevant and related problem reports for the purpose of trend analysis. The complex text syntax in problem descriptions has limited statistical text mining of problem reports. The presentation describes an intelligent tagging approach that applies syntactic and then semantic analysis to overcome this problem. The tags identify types of problems and equipment that are embedded in the text descriptions. The power of these tags is illustrated in a faceted searching and browsing interface for problem report trending that combines automatically generated tags with database code fields and temporal information.

Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

PubMed

Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

2015-10-21

Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments.

Tail proteins of phage T5: investigation of the effect of the His6-tag position, from expression to crystallisation.

PubMed

Noirclerc-Savoye, Marjolaine; Flayhan, Ali; Pereira, Cindy; Gallet, Benoit; Gans, Pierre; Ebel, Christine; Breyton, Cécile

2015-05-01

Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality. Copyright © 2015 Elsevier Inc. All rights reserved.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

PubMed

Wang, Jianhao; Qin, Yuqin; Qin, Haifang; Liu, Li; Ding, Shumin; Teng, Yiwan; Ji, Junling; Qiu, Lin; Jiang, Pengju

2016-08-01

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Laboratory Exercise to Illustrate Protein-Membrane Interactions

ERIC Educational Resources Information Center

Weers, Paul M. M.; Prenner, Elmar J.; Curic, Spomenka; Lohmeier-Vogel, Elke M.

2016-01-01

The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6-tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from pre-dried phospholipid films. When apoA-I is added to the MLV,…

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

PubMed Central

Liu, Wenhao; Howarth, Mark; Greytak, Andrew B.; Zheng, Yi; Nocera, Daniel G.; Ting, Alice Y.; Bawendi, Moungi G.

2009-01-01

We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor’s local chemical environment. PMID:18177042

Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.

PubMed

Twerdochlib, Adriana L; Chubatsu, Leda S; Souza, Emanuel M; Pedrosa, Fábio O; Steffens, M Berenice R; Yates, M Geoffrey; Rigo, Liu U

2003-07-01

NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

PubMed Central

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

Vehicle autonomous localization in local area of coal mine tunnel based on vision sensors and ultrasonic sensors

PubMed Central

Yang, Wei; You, Kaiming; Li, Wei; Kim, Young-il

2017-01-01

This paper presents a vehicle autonomous localization method in local area of coal mine tunnel based on vision sensors and ultrasonic sensors. Barcode tags are deployed in pairs on both sides of the tunnel walls at certain intervals as artificial landmarks. The barcode coding is designed based on UPC-A code. The global coordinates of the upper left inner corner point of the feature frame of each barcode tag deployed in the tunnel are uniquely represented by the barcode. Two on-board vision sensors are used to recognize each pair of barcode tags on both sides of the tunnel walls. The distance between the upper left inner corner point of the feature frame of each barcode tag and the vehicle center point can be determined by using a visual distance projection model. The on-board ultrasonic sensors are used to measure the distance from the vehicle center point to the left side of the tunnel walls. Once the spatial geometric relationship between the barcode tags and the vehicle center point is established, the 3D coordinates of the vehicle center point in the tunnel’s global coordinate system can be calculated. Experiments on a straight corridor and an underground tunnel have shown that the proposed vehicle autonomous localization method is not only able to quickly recognize the barcode tags affixed to the tunnel walls, but also has relatively small average localization errors in the vehicle center point’s plane and vertical coordinates to meet autonomous unmanned vehicle positioning requirements in local area of coal mine tunnel. PMID:28141829

Vehicle autonomous localization in local area of coal mine tunnel based on vision sensors and ultrasonic sensors.

PubMed

Xu, Zirui; Yang, Wei; You, Kaiming; Li, Wei; Kim, Young-Il

2017-01-01

This paper presents a vehicle autonomous localization method in local area of coal mine tunnel based on vision sensors and ultrasonic sensors. Barcode tags are deployed in pairs on both sides of the tunnel walls at certain intervals as artificial landmarks. The barcode coding is designed based on UPC-A code. The global coordinates of the upper left inner corner point of the feature frame of each barcode tag deployed in the tunnel are uniquely represented by the barcode. Two on-board vision sensors are used to recognize each pair of barcode tags on both sides of the tunnel walls. The distance between the upper left inner corner point of the feature frame of each barcode tag and the vehicle center point can be determined by using a visual distance projection model. The on-board ultrasonic sensors are used to measure the distance from the vehicle center point to the left side of the tunnel walls. Once the spatial geometric relationship between the barcode tags and the vehicle center point is established, the 3D coordinates of the vehicle center point in the tunnel's global coordinate system can be calculated. Experiments on a straight corridor and an underground tunnel have shown that the proposed vehicle autonomous localization method is not only able to quickly recognize the barcode tags affixed to the tunnel walls, but also has relatively small average localization errors in the vehicle center point's plane and vertical coordinates to meet autonomous unmanned vehicle positioning requirements in local area of coal mine tunnel.

High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

PubMed

Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

2016-01-01

Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

PubMed

Sarpong, Kwabena; Bose, Ron

2017-03-15

A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

Microparticles: Facile and High-Throughput Synthesis of Functional Microparticles with Quick Response Codes (Small 24/2016).

PubMed

Ramirez, Lisa Marie S; He, Muhan; Mailloux, Shay; George, Justin; Wang, Jun

2016-06-01

Microparticles carrying quick response (QR) barcodes are fabricated by J. Wang and co-workers on page 3259, using a massive coding of dissociated elements (MiCODE) technology. Each microparticle can bear a special custom-designed QR code that enables encryption or tagging with unlimited multiplexity, and the QR code can be easily read by cellphone applications. The utility of MiCODE particles in multiplexed DNA detection and microtagging for anti-counterfeiting is explored. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Effects of Computerized Auditory Feedback on Electronic Article Surveillance Tag Placement in an Auto-Parts Distribution Center

ERIC Educational Resources Information Center

Goomas, David T.

2008-01-01

In this report from the field, computerized auditory feedback was used to inform order selectors and order selector auditors in a distribution center to add an electronic article surveillance (EAS) adhesive tag. This was done by programming handheld computers to emit a loud beep for high-priced items upon scanning the item's bar-coded Universal…

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

PubMed Central

Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

2015-01-01

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

Functionalized Nano-adsorbent for Affinity Separation of Proteins

NASA Astrophysics Data System (ADS)

Zou, Xueyan; Yang, Fengbo; Sun, Xin; Qin, Mingming; Zhao, Yanbao; Zhang, Zhijun

2018-05-01

Thiol-functionalized silica nanospheres (SiO2-SH NSs) with an average diameter of 460 nm were synthesized through a hydrothermal route. Subsequently, the prepared SiO2-SH NSs were modified by SnO2 quantum dots to afford SnO2/SiO2 composite NSs possessing obvious fluorescence, which could be used to trace the target protein. The SnO2/SiO2 NSs were further modified by reduced glutathione (GSH) to obtain SnO2/SiO2-GSH NSs, which can specifically separate glutathione S-transferase-tagged (GST-tagged) protein. Moreover, the peroxidase activity of glutathione peroxidase 3 (GPX3) separated from SnO2/SiO2-GSH NSs in vitro was evaluated. Results show that the prepared SnO2/SiO2-GSH NSs exhibit negligible nonspecific adsorption, high concentration of protein binding (7.4 mg/g), and good reused properties. In the meantime, the GST-tagged GPX3 separated by these NSs can retain its redox state and peroxidase activity. Therefore, the prepared SnO2/SiO2-GSH NSs might find promising application in the rapid separation and purification of GST-tagged proteins.

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

PubMed

Hikone, Yuya; Hirai, Go; Mishima, Masaki; Inomata, Kohsuke; Ikeya, Teppei; Arai, Souichiro; Shirakawa, Masahiro; Sodeoka, Mikiko; Ito, Yutaka

2016-10-01

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Construction of stabilized and tagged foot-and-mouth disease virus.

PubMed

Park, Jeong-Nam; Ko, Mi-Kyeong; Kim, Rae-Hyung; Park, Min-Eun; Lee, Seo-Yong; Yoon, Ji-Eun; Choi, Joo-Hyung; You, Su-Hwa; Park, Jung-Won; Lee, Kwang-Nyeong; Chun, Ji-Eun; Kim, Su-Mi; Tark, Dongseob; Lee, Hyang-Sim; Ko, Young-Joon; Kim, Byounghan; Lee, Myoung-Heon; Park, Jong-Hyeon

2016-11-01

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

Nickel nanoparticle decorated graphene for highly selective isolation of polyhistidine-tagged proteins

NASA Astrophysics Data System (ADS)

Liu, Jia-Wei; Yang, Ting; Ma, Lin-Yu; Chen, Xu-Wei; Wang, Jian-Hua

2013-12-01

Nickel nanoparticle decorated graphene (GP-Ni) is prepared by one-pot hydrothermal reduction of graphene oxide and nickel cations by hydrazine hydrate in the presence of poly(sodium-p-styrenesulfonate) (PSS). The GP-Ni hybrid is characterized by XRD, TEM, SEM, XPS, Raman and FT-IR spectra, demonstrating the formation of poly-dispersed nickel nanoparticles with an average size of 83 nm attached on the surface of graphene sheets. The GP-Ni hybrid exhibits ferromagnetic behavior with a magnetization saturation of 31.1 emu g-1 at 10 000 Oersted (Oe). The GP-Ni also possesses favorable stability in aqueous medium and rapid magnetic response to an external magnetic field. These make it a novel magnetic adsorbent for the separation/isolation of His6-tagged recombinant proteins from a complex sample matrix (cell lysate). The targeted protein species is captured onto the surface of the GP-Ni hybrid via specific metal affinity force between polyhistidine groups and nickel nanoparticles. The SDS-PAGE assay indicates highly selective separation of His6-tagged Smt A from cell lysate. The GP-Ni hybrid displays favorable performance on the separation/isolation of His6-tagged recombinant proteins with respect to the commercial NTA-Ni2+ column.

A New Aloha Anti-Collision Algorithm Based on CDMA

NASA Astrophysics Data System (ADS)

Bai, Enjian; Feng, Zhu

The tags' collision is a common problem in RFID (radio frequency identification) system. The problem has affected the integrity of the data transmission during the process of communication in the RFID system. Based on analysis of the existing anti-collision algorithm, a novel anti-collision algorithm is presented. The new algorithm combines the group dynamic frame slotted Aloha algorithm with code division multiple access technology. The algorithm can effectively reduce the collision probability between tags. Under the same number of tags, the algorithm is effective in reducing the reader recognition time and improve overall system throughput rate.

Printed dose-recording tag based on organic complementary circuits and ferroelectric nonvolatile memories

PubMed Central

Nga Ng, Tse; Schwartz, David E.; Mei, Ping; Krusor, Brent; Kor, Sivkheng; Veres, Janos; Bröms, Per; Eriksson, Torbjörn; Wang, Yong; Hagel, Olle; Karlsson, Christer

2015-01-01

We have demonstrated a printed electronic tag that monitors time-integrated sensor signals and writes to nonvolatile memories for later readout. The tag is additively fabricated on flexible plastic foil and comprises a thermistor divider, complementary organic circuits, and two nonvolatile memory cells. With a supply voltage below 30 V, the threshold temperatures can be tuned between 0 °C and 80 °C. The time-temperature dose measurement is calibrated for minute-scale integration. The two memory bits are sequentially written in a thermometer code to provide an accumulated dose record. PMID:26307438

Wireless SAW passive tag temperature measurement in the collision case

NASA Astrophysics Data System (ADS)

Sorokin, A.; Shepeta, A.; Wattimena, M.

2018-04-01

This paper describes temperature measurement in the multisensor systems based on the radio-frequency identification SAW passive tags which are currently applied in the electric power systems and the switchgears. Different approaches of temperature measurement in the collision case are shown here. The study is based on the tag model with specific topology, which allows us to determine temperature through the response signal with time-frequency information. This research considers the collision case for several passive tags as the temperature sensors which are placed in the switchgear. This research proposal is to analyze the possibility of using several SAW passive sensors in the collision case. We consider the using of the different typical elements for passive surface acoustic wave tag which applies as an anticollision passive sensor. These wireless sensors based on the surface acoustic waves tags contain specifically coded structures. This topology makes possible the reliability of increasing tag identification and the temperature measurement in the collision case. As the results for this case we illustrate simultaneous measurement of at least six sensors.

Analysis of Ethylene Receptor Interactions by Co-immunoprecipitation Assays.

PubMed

Gao, Zhiyong; Schaller, G Eric

2017-01-01

Ethylene receptors are predominantly localized to the endoplasmic reticulum (ER) membrane, and coordinate ethylene signal output through protein-protein interactions with each other and additional signaling components. Here, we describe a co-immunoprecipitation (Co-IP) assay based on the use of the Tandem Affinity Purification (TAP) tag to examine the interactions of ethylene receptors in plant extracts. Human IgG-agarose beads are used to pull down TAP-tagged versions of the protein of interest from detergent extracts of Arabidopsis membranes, and the precipitate then is analyzed immunologically for co-purification of the ethylene receptors. This method has been successfully used to examine interactions of the receptors with each other as well as with the Raf-like kinase CTR1.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.

PubMed

Korshun, Ludmila; Vudmaska, Mariya; Moysa, Larissa; Kovtonjuk, Galina; Mikhalap, Svetlana; Ganova, Larissa; Spivak, Nikolay

2013-12-01

In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo.

PubMed

Roelofs, Anke J; Stewart, Charlotte A; Sun, Shuting; Błażewska, Katarzyna M; Kashemirov, Boris A; McKenna, Charles E; Russell, R Graham G; Rogers, Michael J; Lundy, Mark W; Ebetino, Frank H; Coxon, Fraser P

2012-04-01

Bisphosphonates are widely used antiresorptive drugs that bind to calcium. It has become evident that these drugs have differing affinities for bone mineral; however, it is unclear whether such differences affect their distribution on mineral surfaces. In this study, fluorescent conjugates of risedronate, and its lower-affinity analogues deoxy-risedronate and 3-PEHPC, were used to compare the localization of compounds with differing mineral affinities in vivo. Binding to dentine in vitro confirmed differences in mineral binding between compounds, which was influenced predominantly by the characteristics of the parent compound but also by the choice of fluorescent tag. In growing rats, all compounds preferentially bound to forming endocortical as opposed to resorbing periosteal surfaces in cortical bone, 1 day after administration. At resorbing surfaces, lower-affinity compounds showed preferential binding to resorption lacunae, whereas the highest-affinity compound showed more uniform labeling. At forming surfaces, penetration into the mineralizing osteoid was found to inversely correlate with mineral affinity. These differences in distribution at resorbing and forming surfaces were not observed at quiescent surfaces. Lower-affinity compounds also showed a relatively higher degree of labeling of osteocyte lacunar walls and labeled lacunae deeper within cortical bone, indicating increased penetration of the osteocyte canalicular network. Similar differences in mineralizing surface and osteocyte network penetration between high- and low-affinity compounds were evident 7 days after administration, with fluorescent conjugates at forming surfaces buried under a new layer of bone. Fluorescent compounds were incorporated into these areas of newly formed bone, indicating that "recycling" had occurred, albeit at very low levels. Taken together, these findings indicate that the bone mineral affinity of bisphosphonates is likely to influence their distribution within the skeleton. Copyright © 2012 American Society for Bone and Mineral Research.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

PubMed

Kumada, Yoichi; Otsuki, Ryoko; Sakoda, Yumiko; Akai, Ryota; Matoba, Kazutaka; Katayama, Junko; Kishimoto, Michimasa; Horiuchi, Jun-Ichi

2016-10-20

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips. Copyright © 2016 Elsevier B.V. All rights reserved.

Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches.

PubMed

Lima, Analía; Durán, Rosario; Schujman, Gustavo Enrique; Marchissio, María Julia; Portela, María Magdalena; Obal, Gonzalo; Pritsch, Otto; de Mendoza, Diego; Cerveñansky, Carlos

2011-08-24

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular cloning, expression and characterization of 100K gene of fowl adenovirus-4 for prevention and control of hydropericardium syndrome.

PubMed

Shah, M S; Ashraf, A; Khan, M I; Rahman, M; Habib, M; Qureshi, J A

2016-01-01

Fowl adenovirus-4 is an infectious agent causing Hydropericardium syndrome in chickens. Adenovirus are non-enveloped virions having linear, double stranded DNA. Viral genome codes for few structural and non structural proteins. 100K is an important non-structural viral protein. Open reading frame for coding sequence of 100K protein was cloned with oligo histidine tag and expressed in Escherichia coli as a fusion protein. Nucleotide sequence of the gene revealed that 100K gene of FAdV-4 has high homology (98%) with the respective gene of FAdV-10. Recombinant 100K protein was expressed in E. coli and purified by nickel affinity chromatography. Immunization of chickens with recombinant 100K protein elicited significant serum antibody titers. However challenge protection test revealed that 100K protein conferred little protection (40%) to the immunized chicken against pathogenic viral challenge. So it was concluded that 100K gene has 2397 bp length and recombinant 100K protein has molecular weight of 95 kDa. It was also found that the recombinant protein has little capacity to affect the immune response because in-spite of having an important role in intracellular transport & folding of viral capsid proteins during viral replication, it is not exposed on the surface of the virus at any stage. Copyright © 2015 The International Alliance for Biological Standardization. All rights reserved.

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

PubMed Central

Borchert, Sophie; Czech-Sioli, Manja; Neumann, Friederike; Schmidt, Claudia; Wimmer, Peter; Dobner, Thomas

2014-01-01

ABSTRACT Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential. PMID:24371076

Focused Logistics, Joint Vision 2010: A Joint Logistics Roadmap

DTIC Science & Technology

2010-01-01

AIS). AIT devices include bar codes for individual items, optical memory cards for multipacks and containers, radio frequency tags for containers and...Fortezza Card and Firewall technologies are being developed to prevent unau- thorized access. As for infrastructure, DISA has already made significant in...radio frequency tags and optical memory cards , to continuously update the JTAV database. By September 1998, DSS will be deployed in all wholesale

SNAP dendrimers: multivalent protein display on dendrimer-like DNA for directed evolution.

PubMed

Kaltenbach, Miriam; Stein, Viktor; Hollfelder, Florian

2011-09-19

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Pasek, Marta; Boeggeman, Elizabeth; Ramakrishnan, Boopathy

The expression of recombinant proteins in Escherichia coli often leads to inactive aggregated proteins known as the inclusion bodies. To date, the best available tool has been the use of fusion tags, including the carbohydrate-binding protein; e.g., the maltose-binding protein (MBP) that enhances the solubility of recombinant proteins. However, none of these fusion tags work universally with every partner protein. We hypothesized that galectins, which are also carbohydrate-binding proteins, may help as fusion partners in folding the mammalian proteins in E. coli. Here we show for the first time that a small soluble lectin, human galectin-1, one member of amore » large galectin family, can function as a fusion partner to produce soluble folded recombinant human glycosyltransferase, {beta}-1,4-galactosyltransferase-7 ({beta}4Gal-T7), in E. coli. The enzyme {beta}4Gal-T7 transfers galactose to xylose during the synthesis of the tetrasaccharide linker sequence attached to a Ser residue of proteoglycans. Without a fusion partner, {beta}4Gal-T7 is expressed in E. coli as inclusion bodies. We have designed a new vector construct, pLgals1, from pET-23a that includes the sequence for human galectin-1, followed by the Tev protease cleavage site, a 6x His-coding sequence, and a multi-cloning site where a cloned gene is inserted. After lactose affinity column purification of galectin-1-{beta}4Gal-T7 fusion protein, the unique protease cleavage site allows the protein {beta}4Gal-T7 to be cleaved from galectin-1 that binds and elutes from UDP-agarose column. The eluted protein is enzymatically active, and shows CD spectra comparable to the folded {beta}4Gal-T1. The engineered galectin-1 vector could prove to be a valuable tool for expressing other proteins in E. coli.« less

Evidence that coded-wire-tagging procedures can enhance transmission of Renibacterium salmoninarum in chinook salmon

USGS Publications Warehouse

Elliott, D.G.; Pascho, R.J.

2001-01-01

Binary coded wire tags (CWTs) are used extensively for identification and management of anadromous salmonid populations. A study of bacterial kidney disease (BKD) in two brood year groups of hatchery-reared spring chinook salmon Oncorhynchus tshawytscha provided strong evidence that horizontal transmission of Renibacterium salmoninarum, the causative agent of BKD, might be enhanced by CWT-marking procedures. About 4 months after CWTs were implanted in the snouts of juvenile fish, 14-16 different tissues were sampled from each of 60 fish per brood year group for histological analysis. Of the fish that were positive for R. salmoninarum by histological examination, 41% (7 of 17) of the 1988 brood year fish and 24% (10 of 42) of the 1989 brood year fish had BKD lesions confined to the head near the site of tag implantation. These lesions often resulted in the destruction of tissues of one or both olfactory organs. No focal snout infections were observed in fish that had not been marked with CWTs. Further data obtained from tissue analyses by use of an enzyme-linked immunosorbent assay and a fluorescent antibody test for detection of R. salmoninarum supported the hypothesis that infections of R. salmoninarum can be initiated in the snout tissues of CWT-marked fish and then spread to other organs. The tagging procedures might promote transmission of the pathogen among fish via contaminated tagging needles, by facilitating the entry of pathogens through the injection wound, or both. Limited evidence from this study suggested that implantation of passive integrated transponder tags in the peritoneal cavities of fish might also promote the transmission of R. salmoninarum or exacerbate existing infections. The results indicated a need for strict sanitary procedures during the tagging of fish in populations positive for R. salmoninarum to reduce the probability of enhanced horizontal transmission of the pathogen.

An Analysis of Information Assurance Relating to the Department of Defense Radio Frequency Identification (RFID) Passive Network

DTIC Science & Technology

2005-03-01

codes speed up consumer shopping, package shipping, and inventory tracking. RFID offers many advantages over bar codes, as the table below shows...sunlight” (Accenture, 2001, p. 4). Finally, one of the most significant advantages of RFID is the advent of anti-collision. Anti-collision allows an...RFID reader to read and/or write to multiple tags at one time, which is not possible for bar codes. Despite the many advantages RFID over bar codes

Publicizing Your Web Resources for Maximum Exposure.

ERIC Educational Resources Information Center

Smith, Kerry J.

2001-01-01

Offers advice to librarians for marketing their Web sites on Internet search engines. Advises against relying solely on spiders and recommends adding metadata to the source code and delivering that information directly to the search engines. Gives an overview of metadata and typical coding for meta tags. Includes Web addresses for a number of…

Server-Side Includes Made Simple.

ERIC Educational Resources Information Center

Fagan, Jody Condit

2002-01-01

Describes server-side include (SSI) codes which allow Webmasters to insert content into Web pages without programming knowledge. Explains how to enable the codes on a Web server, provides a step-by-step process for implementing them, discusses tags and syntax errors, and includes examples of their use on the Web site for Southern Illinois…

A review of automatic patient identification options for public health care centers with restricted budgets.

PubMed

García-Betances, Rebeca I; Huerta, Mónica K

2012-01-01

A comparative review is presented of available technologies suitable for automatic reading of patient identification bracelet tags. Existing technologies' backgrounds, characteristics, advantages and disadvantages, are described in relation to their possible use by public health care centers with budgetary limitations. A comparative assessment is presented of suitable automatic identification systems based on graphic codes, both one- (1D) and two-dimensional (2D), printed on labels, as well as those based on radio frequency identification (RFID) tags. The analysis looks at the tradeoffs of these technologies to provide guidance to hospital administrator looking to deploy patient identification technology. The results suggest that affordable automatic patient identification systems can be easily and inexpensively implemented using 2D code printed on low cost bracelet labels, which can then be read and automatically decoded by ordinary mobile smart phones. Because of mobile smart phones' present versatility and ubiquity, the implantation and operation of 2D code, and especially Quick Response® (QR) Code, technology emerges as a very attractive alternative to automate the patients' identification processes in low-budget situations.

A Review of Automatic Patient Identification Options for Public Health Care Centers with Restricted Budgets

PubMed Central

García-Betances, Rebeca I.; Huerta, Mónica K.

2012-01-01

A comparative review is presented of available technologies suitable for automatic reading of patient identification bracelet tags. Existing technologies’ backgrounds, characteristics, advantages and disadvantages, are described in relation to their possible use by public health care centers with budgetary limitations. A comparative assessment is presented of suitable automatic identification systems based on graphic codes, both one- (1D) and two-dimensional (2D), printed on labels, as well as those based on radio frequency identification (RFID) tags. The analysis looks at the tradeoffs of these technologies to provide guidance to hospital administrator looking to deploy patient identification technology. The results suggest that affordable automatic patient identification systems can be easily and inexpensively implemented using 2D code printed on low cost bracelet labels, which can then be read and automatically decoded by ordinary mobile smart phones. Because of mobile smart phones’ present versatility and ubiquity, the implantation and operation of 2D code, and especially Quick Response® (QR) Code, technology emerges as a very attractive alternative to automate the patients’ identification processes in low-budget situations. PMID:23569629

A secure RFID authentication protocol adopting error correction code.

PubMed

Chen, Chien-Ming; Chen, Shuai-Min; Zheng, Xinying; Chen, Pei-Yu; Sun, Hung-Min

2014-01-01

RFID technology has become popular in many applications; however, most of the RFID products lack security related functionality due to the hardware limitation of the low-cost RFID tags. In this paper, we propose a lightweight mutual authentication protocol adopting error correction code for RFID. Besides, we also propose an advanced version of our protocol to provide key updating. Based on the secrecy of shared keys, the reader and the tag can establish a mutual authenticity relationship. Further analysis of the protocol showed that it also satisfies integrity, forward secrecy, anonymity, and untraceability. Compared with other lightweight protocols, the proposed protocol provides stronger resistance to tracing attacks, compromising attacks and replay attacks. We also compare our protocol with previous works in terms of performance.

A Secure RFID Authentication Protocol Adopting Error Correction Code

PubMed Central

Zheng, Xinying; Chen, Pei-Yu

2014-01-01

RFID technology has become popular in many applications; however, most of the RFID products lack security related functionality due to the hardware limitation of the low-cost RFID tags. In this paper, we propose a lightweight mutual authentication protocol adopting error correction code for RFID. Besides, we also propose an advanced version of our protocol to provide key updating. Based on the secrecy of shared keys, the reader and the tag can establish a mutual authenticity relationship. Further analysis of the protocol showed that it also satisfies integrity, forward secrecy, anonymity, and untraceability. Compared with other lightweight protocols, the proposed protocol provides stronger resistance to tracing attacks, compromising attacks and replay attacks. We also compare our protocol with previous works in terms of performance. PMID:24959619

Identification of midgut proteins that are differentially expressed in trypanosome-susceptible and normal tsetse flies (Glossina morsitans morsitans).

PubMed

Haddow, J D; Haines, L R; Gooding, R H; Olafson, R W; Pearson, T W

2005-05-01

Molecules in the midgut of tsetse flies (Diptera: Glossinidiae) are thought to play important roles in the life cycle of African trypanosomes by influencing initial parasite establishment and subsequent differentiation events that ultimately lead to maturation of mammal-infective trypanosomes. The molecular composition of the tsetse midgut is, therefore, of critical importance to disease transmission by these medically important vectors. In this study we compared protein expression profiles of midguts of the salmon mutant and wild type Glossina morsitans morsitans Westwood that display marked differences in their susceptibility to infection by African trypanosomes. Isotope coded affinity tag (ICAT) technology was used to identify 207 proteins including 17 that were up regulated and nine that were down regulated in the salmon mutants. Several of the up regulated molecules were previously described as tsetse midgut or salivary gland proteins. Of particular interest was the up regulation in the salmon flies of tsetse midgut EP protein, a recently described molecule with lectin-like activity that was also found to be induced in tsetse by bacterial challenge. The up regulation of the EP protein in midguts of salmon mutants was confirmed by two-dimensional gel electrophoresis and tandem mass spectrometry.

Expression and purification of a functional recombinant aspartate aminotransferase (AST) from Escherichia coli.

PubMed

Zou, Lihui; Zhao, Haijian; Wang, Daguang; Wang, Meng; Zhang, Chuanbao; Xiao, Fei

2014-07-01

Aspartate aminotransferase (AST; E.C. 2.6.1.1), a vitamin B6-dependent enzyme, preferentially promotes the mutual transformation of aspartate and α-ketoglutarate to oxaloacetate and glutamate. It plays a key role in amino acid metabolism and has been widely recommended as a biomarker of liver and heart damage. Our study aimed to evaluate the extensive preparation of AST and its application in quality control in clinical laboratories. We describe a scheme to express and purify the 6His-AST fusion protein. An optimized sequence coding AST was synthesized and transformed into Escherichia coli BL21 (DE3) strain for protein expression. Ideally, the fusion protein has a volumetric productivity achieving 900 mg/l cultures. After affinity chromatography, the enzyme activity of purified AST reached 150,000 U/L. Commutability assessment between the engineered AST and standard AST from Roche suggested that the engineered AST was the better candidate for the reference material. Moreover, the AST showed high stability during long-term storage at -20ºC. In conclusion, the highly soluble 6His-tagged AST can become a convenient tool for supplying a much better and cheaper standard or reference material for the clinical laboratory.

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

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

Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.

2010-01-01

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, andmore » enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.« less

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

PubMed

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

PubMed Central

Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

2015-01-01

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

Mass spectrometry-based methods for detection and differentiation of botulinum neurotoxins

DOEpatents

Schmidt, Jurgen G [Los Alamos, NM; Boyer, Anne E [Atlanta, GA; Kalb, Suzanne R [Atlanta, GA; Moura, Hercules [Tucker, GA; Barr, John R [Suwannee, GA; Woolfitt, Adrian R [Atlanta, GA

2009-11-03

The present invention is directed to a method for detecting the presence of clostridial neurotoxins in a sample by mixing a sample with a peptide that can serve as a substrate for proteolytic activity of a clostridial neurotoxin; and measuring for proteolytic activity of a clostridial neurotoxin by a mass spectroscopy technique. In one embodiment, the peptide can have an affinity tag attached at two or more sites.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

PubMed

Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

2015-02-23

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

GLUT-3 expression in human skeletal muscle

NASA Technical Reports Server (NTRS)

Stuart, C. A.; Wen, G.; Peng, B. H.; Popov, V. L.; Hudnall, S. D.; Campbell, G. A.

2000-01-01

Muscle biopsy homogenates contain GLUT-3 mRNA and protein. Before these studies, it was unclear where GLUT-3 was located in muscle tissue. In situ hybridization using a midmolecule probe demonstrated GLUT-3 within all muscle fibers. Fluorescent-tagged antibody reacting with affinity-purified antibody directed at the carboxy-terminus demonstrated GLUT-3 protein in all fibers. Slow-twitch muscle fibers, identified by NADH-tetrazolium reductase staining, possessed more GLUT-3 protein than fast-twitch fibers. Electron microscopy using affinity-purified primary antibody and gold particle-tagged second antibody showed that the majority of GLUT-3 was in association with triads and transverse tubules inside the fiber. Strong GLUT-3 signals were seen in association with the few nerves that traversed muscle sections. Electron microscopic evaluation of human peripheral nerve demonstrated GLUT-3 within the axon, with many of the particles related to mitochondria. GLUT-3 protein was found in myelin but not in Schwann cells. GLUT-1 protein was not present in nerve cells, axons, myelin, or Schwann cells but was seen at the surface of the peripheral nerve in the perineurium. These studies demonstrated that GLUT-3 mRNA and protein are expressed throughout normal human skeletal muscle, but the protein is predominantly found in the triads of slow-twitch muscle fibers.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions

PubMed Central

Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

2015-01-01

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine “cloning scar” present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected. PMID:25704442

Graphene Field-Effect Transistors for the Sensitive and Selective Detection of Escherichia coli Using Pyrene-Tagged DNA Aptamer.

PubMed

Wu, Guangfu; Dai, Ziwen; Tang, Xin; Lin, Zihong; Lo, Pik Kwan; Meyyappan, M; Lai, King Wai Chiu

2017-10-01

This study reports biosensing using graphene field-effect transistors with the aid of pyrene-tagged DNA aptamers, which exhibit excellent selectivity, affinity, and stability for Escherichia coli (E. coli) detection. The aptamer is employed as the sensing probe due to its advantages such as high stability and high affinity toward small molecules and even whole cells. The change of the carrier density in the probe-modified graphene due to the attachment of E. coli is discussed theoretically for the first time and also verified experimentally. The conformational change of the aptamer due to the binding of E. coli brings the negatively charged E. coli close to the graphene surface, increasing the hole carrier density efficiently in graphene and achieving electrical detection. The binding of negatively charged E. coli induces holes in graphene, which are pumped into the graphene channel from the contact electrodes. The carrier mobility, which correlates the gate voltage to the electrical signal of the APG-FETs, is analyzed and optimized here. The excellent sensing performance such as low detection limit, high sensitivity, outstanding selectivity and stability of the graphene biosensor for E. coli detection paves the way to develop graphene biosensors for bacterial detection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of coded wire tags for marking lake trout

USGS Publications Warehouse

Elrod, Joseph H.; Schneider, Clifford P.

1986-01-01

Among hatchery-reared lake trout (Salvelinus namaycush) of the 1979-1982 year classes stocked in New York waters of Lake Ontario, more than 3 million fish were marked with a coded wire tag (CWT) plus an adipose fin clip, and 1.5 million with only conventional fin clips. Altogether, 7,640 tags were recovered from fish collected with bottom trawls and gill nets or caught by anglers during 1980-1983. One person was able to extract and decipher 200 or more CWTs per day with about a 1% error rate in reading and recording codes. Presence of the CWT did not affect growth. The adipose fin clip did not regenerate. The occurrence of fish with an adipose fin clip but no CWT resulted primarily from the regeneration of paired fins among fish marked with a combination of the adipose fin and a paired fin. Loss of CWTs between marking and stocking (generally 4-5 months for fish stocked in spring and 1-8 d for fish stocked in fall) declined from nearly 11% for the 1979 year class stocked as fall fingerlings to less than 3% for the 1981 and 1982 year classes - a difference that primarily reflected improvements in instrumentation and tagging technique. The rate of CWT loss after the marked fish were stocked was probably less than 1% per year. The CWT is a reliable method for marking hatchery-reared lake trout. A large number of experimental groups can be uniquely marked, and fish from each group can be accurately identified throughout their life. Use of this technique should greatly facilitate evaluations of genetic strain, hatchery experience, condition at time of stocking, season of stocking, size at stocking, method of stocking and other factors that affect poststocking survival and performance of lake trout stocked in the Great Lakes.

Synthesis of poly(N-isopropylacrylamide) particles for metal affinity binding of peptides

PubMed Central

Tsai, Hsin-Yi; Lee, Alexander; Peng, Wei; Yates, Matthew Z.

2013-01-01

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgel particles with metal affinity ligands were prepared for selective binding of peptides containing the His6-tag (six consecutive histidine residues). The PNIPAM particles were copolymerized with the functional ligand vinylbenzyl iminodiacetic acid (VBIDA) through a two-stage dispersion polymerization using poly(N-vinyl pyrrolidone) (PVP) as a steric stabilizer. The resulting particles were monodisperse in size and colloidally stable over a wide range of temperature and ionic strength due to chemically grafted PVP chains. The particle size was also found to be sensitive to ionic strength and pH of the aqueous environment, likely due to the electrostatic repulsion between ionized VBIDA groups. Divalent nickel ions were chelated to the VBIDA groups, allowing selective metal affinity attachment of a His6-Cys peptide. The peptide was released upon the addition of the competitive ligand imidazole, demonstrating that the peptide attachment to the particles is reversible and selective. PMID:24176889

Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

NASA Astrophysics Data System (ADS)

Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

2017-07-01

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

Evaluation of IDA-PEVA hollow fiber membrane metal ion affinity chromatography for purification of a histidine-tagged human proinsulin.

PubMed

de Aquino, Luciana Cristina Lins; de Sousa, Heloisa Ribeiro Tunes; Miranda, Everson Alves; Vilela, Luciano; Bueno, Sônia Maria Alves

2006-04-13

Inabilities to process particulate material and to allow the use of high flow rates are limitations of conventional chromatography. Membranes have been suggested as matrix for affinity separation due to advantages such as allowing high flow rates and low-pressure drops. This work evaluated the feasibility of using an iminodiacetic acid linked poly(ethylenevinyl alcohol) membrane in the immobilized metal ion affinity chromatography (IMAC) purification of a human proinsulin(His)(6) of an industrial insulin production process. The screening of metal ions showed Ni(2+) as metal with higher selectivity and capacity among the Cu(2+), Ni(2+), Zn(2+) and Co(2+). The membrane showed to be equivalent to conventional chelating beads in terms of selectivity and had a lower capacity (3.68 mg/g versus 12.26 mg/g). The dynamic adsorption capacity for human proinsulin(His)(6) was unaffected by the mode of operation (dead-end and cross-flow filtration).

A genome-wide resource for the analysis of protein localisation in Drosophila

PubMed Central

Sarov, Mihail; Barz, Christiane; Jambor, Helena; Hein, Marco Y; Schmied, Christopher; Suchold, Dana; Stender, Bettina; Janosch, Stephan; KJ, Vinay Vikas; Krishnan, RT; Krishnamoorthy, Aishwarya; Ferreira, Irene RS; Ejsmont, Radoslaw K; Finkl, Katja; Hasse, Susanne; Kämpfer, Philipp; Plewka, Nicole; Vinis, Elisabeth; Schloissnig, Siegfried; Knust, Elisabeth; Hartenstein, Volker; Mann, Matthias; Ramaswami, Mani; VijayRaghavan, K; Tomancak, Pavel; Schnorrer, Frank

2016-01-01

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts. DOI: http://dx.doi.org/10.7554/eLife.12068.001 PMID:26896675

Radar transponder apparatus and signal processing technique

DOEpatents

Axline, Jr., Robert M.; Sloan, George R.; Spalding, Richard E.

1996-01-01

An active, phase-coded, time-grating transponder and a synthetic-aperture radar (SAR) and signal processor means, in combination, allow the recognition and location of the transponder (tag) in the SAR image and allow communication of information messages from the transponder to the SAR. The SAR is an illuminating radar having special processing modifications in an image-formation processor to receive an echo from a remote transponder, after the transponder receives and retransmits the SAR illuminations, and to enhance the transponder's echo relative to surrounding ground clutter by recognizing special transponder modulations from phase-shifted from the transponder retransmissions. The remote radio-frequency tag also transmits information to the SAR through a single antenna that also serves to receive the SAR illuminations. Unique tag-modulation and SAR signal processing techniques, in combination, allow the detection and precise geographical location of the tag through the reduction of interfering signals from ground clutter, and allow communication of environmental and status information from said tag to be communicated to said SAR.

Radar transponder apparatus and signal processing technique

DOEpatents

Axline, R.M. Jr.; Sloan, G.R.; Spalding, R.E.

1996-01-23

An active, phase-coded, time-grating transponder and a synthetic-aperture radar (SAR) and signal processor means, in combination, allow the recognition and location of the transponder (tag) in the SAR image and allow communication of information messages from the transponder to the SAR. The SAR is an illuminating radar having special processing modifications in an image-formation processor to receive an echo from a remote transponder, after the transponder receives and retransmits the SAR illuminations, and to enhance the transponder`s echo relative to surrounding ground clutter by recognizing special transponder modulations from phase-shifted from the transponder retransmissions. The remote radio-frequency tag also transmits information to the SAR through a single antenna that also serves to receive the SAR illuminations. Unique tag-modulation and SAR signal processing techniques, in combination, allow the detection and precise geographical location of the tag through the reduction of interfering signals from ground clutter, and allow communication of environmental and status information from said tag to be communicated to said SAR. 4 figs.

Nassi-Schneiderman Diagram in HTML Based on AML

ERIC Educational Resources Information Center

Menyhárt, László

2013-01-01

In an earlier work I defined an extension of XML called Algorithm Markup Language (AML) for easy and understandable coding in an IDE which supports XML editing (e.g. NetBeans). The AML extension contains annotations and native language (English or Hungarian) tag names used when coding our algorithm. This paper presents a drawing tool with which…

Recombinant expression of Intrepicalcin from the scorpion Vaejovis intrepidus and its effect on skeletal ryanodine receptors.

PubMed

Vargas-Jaimes, Leonel; Xiao, Liang; Zhang, Jing; Possani, Lourival D; Valdivia, Héctor H; Quintero-Hernández, Verónica

2017-04-01

Scorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. Recombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. [ 3 H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with K d =17.4±4.0nM. Intrepicalcin significantly augments the bell-shaped [Ca 2+ ]-[ 3 H]ryanodine binding curve at all [Ca 2+ ] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca 2+ release at an initial dose=45.3±2.5nM, and depletes ~50% of Ca 2+ load from skeletal sarcoplasmic reticulum vesicles. We conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. This is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

PubMed Central

Fujishiro, Mitsuhiro; Okamura, Tomohisa; Fujio, Keishi; Okazaki, Hiroaki; Nomura, Seitaro; Takeda, Norifumi; Harada, Mutsuo; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Yamamoto, Kazuhiko; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays. PMID:26020647

Identification of novel lysosomal matrix proteins by proteome analysis.

PubMed

Kollmann, Katrin; Mutenda, Kudzai E; Balleininger, Martina; Eckermann, Ellen; von Figura, Kurt; Schmidt, Bernhard; Lübke, Torben

2005-10-01

The lysosomal matrix is estimated to contain about 50 different proteins. Most of the matrix proteins are acid hydrolases that depend on mannose 6-phosphate receptors (MPR) for targeting to lysosomes. Here, we describe a comprehensive proteome analysis of MPR-binding proteins from mouse. Mouse embryonic fibroblasts defective in both MPR (MPR 46-/- and MPR 300-/-) are known to secrete the lysosomal matrix proteins. Secretions of these cells were affinity purified using an affinity matrix derivatized with MPR46 and MPR300. In the protein fraction bound to the affinity matrix and eluted with mannose 6-phosphate, 34 known lysosomal matrix proteins, 4 candidate proteins of the lysosomal matrix and 4 non-lysosomal contaminants were identified by mass spectrometry after separation by two-dimensional gel electrophoresis or by multidimensional protein identification technology. For 3 of the candidate proteins, mammalian ependymin-related protein-2 (MERP-2), retinoid-inducible serine carboxypeptidase (RISC) and the hypothetical 66.3-kDa protein we could verify that C-terminally tagged forms bound in an M6P-dependent manner to an MPR-affinity matrix and were internalized via MPR-mediated endocytosis. Hence these 3 proteins are likely to represent hitherto unrecognized lysosomal matrix proteins.

A rapid and fluorogenic TMP-AcBOPDIPY probe for covalent labeling of proteins in live cells.

PubMed

Liu, Wei; Li, Fu; Chen, Xi; Hou, Jian; Yi, Long; Wu, Yao-Wen

2014-03-26

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

A Suite of "Minimalist" Photo-Crosslinkers for Live-Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors.

PubMed

Pan, Sijun; Jang, Se-Young; Wang, Danyang; Liew, Si Si; Li, Zhengqiu; Lee, Jun-Seok; Yao, Shao Q

2017-09-18

Affinity-based probes (AfBPs) provide a powerful tool for large-scale chemoproteomic studies of drug-target interactions. The development of high-quality probes capable of recapitulating genuine drug-target engagement, however, could be challenging. "Minimalist" photo-crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine-containing photo-crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of AfBPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug-target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC-based chemoproteomic studies have led to the discovery of a novel off-target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up-regulation of endogenous DNMT1 expression under normoxia conditions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biochemical localization of a protein involved in Gluconacetobacter hansenii cellulose synthesis

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

Iyer, Prashanti R; Catchmark, Jeffrey M; Brown, Nicole Robitaille

2011-02-08

Using subcellular fractionation and Western blot methods, we have shown that AcsD, one of the proteins encoded by the Acetobacter cellulose synthase (acs) operon, is localized in the periplasmic region of the cell. AcsD protein was heterologously expressed in Escherichia coli and purified using histidine tag affinity methods. The purified protein was used to obtain rabbit polyclonal antibodies. The purity of the subcellular fractions was assessed by marker enzyme assays.

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag

PubMed Central

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant. PMID:26539718

Use of the Nanofitin Alternative Scaffold as a GFP-Ready Fusion Tag.

PubMed

Huet, Simon; Gorre, Harmony; Perrocheau, Anaëlle; Picot, Justine; Cinier, Mathieu

2015-01-01

With the continuous diversification of recombinant DNA technologies, the possibilities for new tailor-made protein engineering have extended on an on-going basis. Among these strategies, the use of the green fluorescent protein (GFP) as a fusion domain has been widely adopted for cellular imaging and protein localization. Following the lead of the direct head-to-tail fusion of GFP, we proposed to provide additional features to recombinant proteins by genetic fusion of artificially derived binders. Thus, we reported a GFP-ready fusion tag consisting of a small and robust fusion-friendly anti-GFP Nanofitin binding domain as a proof-of-concept. While limiting steric effects on the carrier, the GFP-ready tag allows the capture of GFP or its blue (BFP), cyan (CFP) and yellow (YFP) alternatives. Here, we described the generation of the GFP-ready tag from the selection of a Nanofitin variant binding to the GFP and its spectral variants with a nanomolar affinity, while displaying a remarkable folding stability, as demonstrated by its full resistance upon thermal sterilization process or the full chemical synthesis of Nanofitins. To illustrate the potential of the Nanofitin-based tag as a fusion partner, we compared the expression level in Escherichia coli and activity profile of recombinant human tumor necrosis factor alpha (TNFα) constructs, fused to a SUMO or GFP-ready tag. Very similar expression levels were found with the two fusion technologies. Both domains of the GFP-ready tagged TNFα were proved fully active in ELISA and interferometry binding assays, allowing the simultaneous capture by an anti-TNFα antibody and binding to the GFP, and its spectral mutants. The GFP-ready tag was also shown inert in a L929 cell based assay, demonstrating the potent TNFα mediated apoptosis induction by the GFP-ready tagged TNFα. Eventually, we proposed the GFP-ready tag as a versatile capture and labeling system in addition to expected applications of anti-GFP Nanofitins (as illustrated with previously described state-of-the-art anti-GFP binders applied to living cells and in vitro applications). Through a single fusion domain, the GFP-ready tagged proteins benefit from subsequent customization within a wide range of fluorescence spectra upon indirect binding of a chosen GFP variant.

HEC Applications on Columbia Project

NASA Technical Reports Server (NTRS)

Taft, Jim

2004-01-01

NASA's Columbia system consists of a cluster of twenty 512 processor SGI Altix systems. Each of these systems is 3 TFLOP/s in peak performance - approximately the same as the entire compute capability at NAS just one year ago. Each 512p system is a single system image machine with one Linunx O5, one high performance file system, and one globally shared memory. The NAS Terascale Applications Group (TAG) is chartered to assist in scaling NASA's mission critical codes to at least 512p in order to significantly improve emergency response during flight operations, as well as provide significant improvements in the codes. and rate of scientific discovery across the scientifc disciplines within NASA's Missions. Recent accomplishments are 4x improvements to codes in the ocean modeling community, 10x performance improvements in a number of computational fluid dynamics codes used in aero-vehicle design, and 5x improvements in a number of space science codes dealing in extreme physics. The TAG group will continue its scaling work to 2048p and beyond (10240 cpus) as the Columbia system becomes fully operational and the upgrades to the SGI NUMAlink memory fabric are in place. The NUMlink uprades dramatically improve system scalability for a single application. These upgrades will allow a number of codes to execute faster at higher fidelity than ever before on any other system, thus increasing the rate of scientific discovery even further

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

PubMed

Nilsson, J; Bosnes, M; Larsen, F; Nygren, P A; Uhlén, M; Lundeberg, J

1997-04-01

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

Identification of Small RNA-Protein Partners in Plant Symbiotic Bacteria.

PubMed

Robledo, Marta; Matia-González, Ana M; García-Tomsig, Natalia I; Jiménez-Zurdo, José I

2018-01-01

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin. As proof of principle, we show recovery of the RNA chaperone Hfq associated to the strictly Hfq-dependent AbcR2 trans-sRNA. This method can be applied for the investigation of sRNA-protein interactions on a broad range of genetically tractable α-proteobacteria.

UHF wearable battery free sensor module for activity and falling detection.

PubMed

Nam Trung Dang; Thang Viet Tran; Wan-Young Chung

2016-08-01

Falling is one of the most serious medical and social problems in aging population. Therefore taking care of the elderly by detecting activity and falling for preventing and mitigating the injuries caused by falls needs to be concerned. This study proposes a wearable, wireless, battery free ultra-high frequency (UHF) smart sensor tag module for falling and activity detection. The proposed tag is powered by UHF RF wave from reader and read by a standard UHF Electronic Product Code (EPC) Class-1 Generation-2 reader. The battery free sensor module could improve the wearability of the wireless device. The combination of accelerometer signal and received signal strength indication (RSSI) from a reader in the passive smart sensor tag detect the activity and falling of the elderly very successfully. The fabricated smart sensor tag module has an operating range of up to 2.5m and conducting in real-time activity and falling detection.

Decoration of silicon nanostructures with copper particles for simultaneous selective capture and mass spectrometry detection of His-tagged model peptide.

PubMed

Coffinier, Yannick; Kurylo, Ievgen; Drobecq, Hervé; Szunerits, Sabine; Melnyk, Oleg; Zaitsev, Vladimir N; Boukherroub, Rabah

2014-10-21

We present in this work a simple and fast preparation method of a new affinity surface-assisted laser/desorption ionization mass spectrometry (SALDI-MS) substrate based on silicon nanostructures decorated with copper particles. The silicon nanostructures were fabricated by the metal-assisted chemical etching (MACE) method. Then, superhydrophilic areas surrounded by superhydrophobic regions were formed through hydrosilylation reaction of 1-octadecene, followed by local degradation of the octadecyl layer. After that, copper particles were deposited in the hydrophilic areas by using the electroless method. We have demonstrated that these surfaces were able to perform high selective capture of model His-tag peptide even in a complex mixture such as serum solution. Then, the captured peptide was detected by mass spectrometry at a femtomolar level without the need of organic matrix.

Lower Granite Dam Smolt Monitoring Program, 2003-2004 Annual Report.

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

Mensik, Fred; Rapp, Shawn; Ross, Doug

2004-08-01

The 2003 fish collection season at Lower Granite Dam Juvenile Fish Facility (LGR) was characterized by water temperatures, total flows and spill that were below the five year average, low levels of debris, and increased smolt collection numbers compared to 2002 with the exception of unclipped sockeye/kokanee. There were 6,183,825 juvenile salmonids collected. Of these, 6,054,167 were transported to release sites below Bonneville Dam, 5,957,885 by barge and 96,282 by truck. An additional 102,340 fish were bypassed back to the river, primarily due to research projects with another 62,122 bypassed through the PIT-tag bypass system. According to the PTAGIS database,more » 152,268 PIT-tagged fish were detected at Lower Granite Dam. Of these, Smolt Monitoring Staff recorded 345 PIT-tagged raceway and sample mortalities. Of the 6,183,825 total fish collected, 113,290 were PIT-tagged or radio tagged and 380 were sacrificed by researchers. The collection included 836,885 fish that had hatchery marks other than clipped fins (elastomer, freeze brands or Coded Wire Tags). An estimated 54,857 incidental fish were collected with an additional 8,730 adult salmonids removed from the separator.« less

Regulation of the catalytic behavior of pullulanases chelated onto nickel (II)-modified magnetic nanoparticles.

PubMed

Wang, Jianfeng; Liu, Zhongmei; Zhou, Zhemin

2017-06-01

Chelating of pullulanases onto nickel (II)-modified magnetic nanoparticles results in one-step purification and immobilization of pullulanase, and facilitates the commercial application of pullulanase in industrial scale. To improve the catalytic behavior, especially the operational stability, of the nanocatalyst in consecutive batch reactions, we prepared various iminodiacetic acid-modified magnetic nanoparticles differed in surface polarity and spacer length, on which the His6-tagged pullulanases were chelated via nickel ions, and then studied the correlation between the MNPs surface property and the corresponding catalyst behavior. When pullulanases were chelated onto the surface-modified MNPs, the thermostability of all pullulanase derivatives were lower than that of free counterpart, being not relevant to the protein orientation guided by the locality of the His6-tag, but related to the MNPs basal surface polarity and the grafted spacer length. After chelating of pullulanases onto MNPs, there were changes observed in the pH-activity profile and the apparent Michaelis constant toward pullulan. The changing tendencies were mainly dependent on the His6-tagged pullulanase orientation, and the changing extents were tuned by the spacer length. The reusability of pullulanase immobilized by N-terminal His6-tag was higher than that of pullulanase immobilized by C-terminal His6-tag. Moreover, the reusability of the immobilized pullulanase tested increased till grafting polyether amine-400 as spacer-arm, therefore the N-terminal His6-tagged pullulanase chelating MNPs grafted polyether amine-400 gave the best reusability, which retained 60% of initial activity after 18 consecutive cycles with a total reaction time of 9h. Additionally, the correlation analysis of the catalyst behaviors indicated that the reusability was independent from other catalytic properties such as thermostability and substrate affinity. All the results revealed that the catalyst behavior can be mainly controlled by the His6-tagged pullulanase orientation than by the MNPs surface property which can tune the catalyst function. Copyright © 2017. Published by Elsevier Inc.

I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions.

PubMed

Tackett, Alan J; DeGrasse, Jeffrey A; Sekedat, Matthew D; Oeffinger, Marlene; Rout, Michael P; Chait, Brian T

2005-01-01

Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of nonspecifically interacting proteins. We describe a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. I-DIRT will be of broad value for studying protein complexes in biological systems that can be metabolically labeled.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of Deinococcus radiodurans thioredoxin reductase

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

Obiero, Josiah; Bonderoff, Sara A.; Goertzen, Meghan M.

2006-08-01

Recombinant D. radiodurans TrxR with a His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. Deinococcus radiodurans, a Gram-positive bacterium capable of withstanding extreme ionizing radiation, contains two thioredoxins (Trx and Trx1) and a single thioredoxin reductase (TrxR) as part of its response to oxidative stress. Thioredoxin reductase is a member of the family of pyridine nucleotide-disulfide oxidoreductase flavoenzymes. Recombinant D. radiodurans TrxR with amore » His tag at the N-terminus was expressed in Escherichia coli and purified by metal-affinity chromatography. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of 35% PEG 4000, 0.2 M ammonium acetate and citric acid buffer pH 5.1 at 293 K. X-ray diffraction data were collected on a cryocooled crystal to a resolution of 1.9 Å using a synchrotron-radiation source. The space group was determined to be P3{sub 2}21, with unit-cell parameters a = b = 84.33, c = 159.88 Å. The structure of the enzyme has been solved by molecular-replacement methods and structure refinement is in progress.« less

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

PubMed

Guillén-Chable, Francisco; Arenas-Sosa, Iván; Islas-Flores, Ignacio; Corzo, Gerardo; Martinez-Liu, Cynthia; Estrada, Georgina

2017-08-01

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

PubMed

Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

2017-05-28

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

PubMed Central

Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

PubMed

Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

2016-01-01

Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

PubMed Central

Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-01

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

PubMed Central

Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro

2016-01-01

Abstract Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies. PMID:27595817

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad Javad; Forouzandeh-Moghadam, Mehdi; Allameh, Abdol-Amir

2005-11-01

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

PubMed

Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

2014-01-15

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Image Location Estimation by Salient Region Matching.

PubMed

Qian, Xueming; Zhao, Yisi; Han, Junwei

2015-11-01

Nowadays, locations of images have been widely used in many application scenarios for large geo-tagged image corpora. As to images which are not geographically tagged, we estimate their locations with the help of the large geo-tagged image set by content-based image retrieval. In this paper, we exploit spatial information of useful visual words to improve image location estimation (or content-based image retrieval performances). We proposed to generate visual word groups by mean-shift clustering. To improve the retrieval performance, spatial constraint is utilized to code the relative position of visual words. We proposed to generate a position descriptor for each visual word and build fast indexing structure for visual word groups. Experiments show the effectiveness of our proposed approach.

Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.

PubMed

Gupta, Neha; Shrestha, Abhinav; Panda, Amulya Kumar; Gupta, Satish Kumar

2013-07-01

Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.

Identification of Crosslinked Peptides after Click-based Enrichment Using Sequential CID and ETD Tandem Mass Spectrometry

PubMed Central

Chowdhury, Saiful M.; Du, Xiuxia; Tolić, Nikola; Wu, Si; Moore, Ronald J.; Mayer, M. Uljana; Smith, Richard D.; Adkins, Joshua N.

2010-01-01

Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO2-) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO2- moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Click-enabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides. PMID:19496583

Studying Nuclear Receptor Complexes in the Cellular Environment.

PubMed

Schaufele, Fred

2016-01-01

The ligand-regulated structure and biochemistry of nuclear receptor complexes are commonly determined by in vitro studies of isolated receptors, cofactors, and their fragments. However, in the living cell, the complexes that form are governed not just by the relative affinities of isolated cofactors for the receptor but also by the cell-specific sequestration or concentration of subsets of competing or cooperating cofactors, receptors, and other effectors into distinct subcellular domains and/or their temporary diversion into other cellular activities. Most methods developed to understand nuclear receptor function in the cellular environment involve the direct tagging of the nuclear receptor or its cofactors with fluorescent proteins (FPs) and the tracking of those FP-tagged factors by fluorescence microscopy. One of those approaches, Förster resonance energy transfer (FRET) microscopy, quantifies the transfer of energy from a higher energy "donor" FP to a lower energy "acceptor" FP attached to a single protein or to interacting proteins. The amount of FRET is influenced by the ligand-induced changes in the proximities and orientations of the FPs within the tagged nuclear receptor complexes, which is an indicator of the structure of the complexes, and by the kinetics of the interaction between FP-tagged factors. Here, we provide a guide for parsing information about the structure and biochemistry of nuclear receptor complexes from FRET measurements in living cells.

Experimental studies related to the origin of the genetic code and the process of protein synthesis - A review

NASA Technical Reports Server (NTRS)

Lacey, J. C., Jr.; Mullins, D. W., Jr.

1983-01-01

A survey is presented of the literature on the experimental evidence for the genetic code assignments and the chemical reactions involved in the process of protein synthesis. In view of the enormous number of theoretical models that have been advanced to explain the origin of the genetic code, attention is confined to experimental studies. Since genetic coding has significance only within the context of protein synthesis, it is believed that the problem of the origin of the code must be dealt with in terms of the origin of the process of protein synthesis. It is contended that the answers must lie in the nature of the molecules, amino acids and nucleotides, the affinities they might have for one another, and the effect that those affinities must have on the chemical reactions that are related to primitive protein synthesis. The survey establishes that for the bulk of amino acids, there is a direct and significant correlation between the hydrophobicity rank of the amino acids and the hydrophobicity rank of their anticodonic dinucleotides.

Carbon source-dependent expansion of the genetic code in bacteria

PubMed Central

Prat, Laure; Heinemann, Ilka U.; Aerni, Hans R.; Rinehart, Jesse; O’Donoghue, Patrick; Söll, Dieter

2012-01-01

Despite the fact that the genetic code is known to vary between organisms in rare cases, it is believed that in the lifetime of a single cell the code is stable. We found Acetohalobium arabaticum cells grown on pyruvate genetically encode 20 amino acids, but in the presence of trimethylamine (TMA), A. arabaticum dynamically expands its genetic code to 21 amino acids including pyrrolysine (Pyl). A. arabaticum is the only known organism that modulates the size of its genetic code in response to its environment and energy source. The gene cassette pylTSBCD, required to biosynthesize and genetically encode UAG codons as Pyl, is present in the genomes of 24 anaerobic archaea and bacteria. Unlike archaeal Pyl-decoding organisms that constitutively encode Pyl, we observed that A. arabaticum controls Pyl encoding by down-regulating transcription of the entire Pyl operon under growth conditions lacking TMA, to the point where no detectable Pyl-tRNAPyl is made in vivo. Pyl-decoding archaea adapted to an expanded genetic code by minimizing TAG codon frequency to typically ∼5% of ORFs, whereas Pyl-decoding bacteria (∼20% of ORFs contain in-frame TAGs) regulate Pyl-tRNAPyl formation and translation of UAG by transcriptional deactivation of genes in the Pyl operon. We further demonstrate that Pyl encoding occurs in a bacterium that naturally encodes the Pyl operon, and identified Pyl residues by mass spectrometry in A. arabaticum proteins including two methylamine methyltransferases. PMID:23185002

High data density and capacity in chipless radiofrequency identification (chipless-RFID) tags based on double-chains of S-shaped split ring resonators (S-SRRs)

NASA Astrophysics Data System (ADS)

Herrojo, Cristian; Mata-Contreras, Javier; Paredes, Ferran; Martín, Ferran

2017-11-01

The data density per surface (DPS) is a figure of merit in chipless radiofrequency identification (chipless-RFID) tags. In this paper, it is demonstrated that chipless-RFID tags with high DPS can be implemented by using double-chains of S-shaped split ring resonators (S-SRRs). Tag reading is achieved by near-field coupling between the tag and the reader, a CPW transmission line fed by a harmonic signal tuned to the resonance frequency of the S-SRRs. By transversally displacing the tag over the CPW, the transmission coefficient of the line is modulated by tag motion. This effectively modulates the amplitude of the injected (carrier) signal at the output port of the line, and the identification (ID) code, determined by the presence or absence of S-SRRs at predefined and equidistant positions in the chains, is contained in the envelope function. The DPS is determined by S-SRR dimensions and by the distance between S-SRRs in the chains. However, by using two chains of S-SRRs, the number of bits per unit length that can be accommodated is very high. This chipless-RFID system is of special interest in applications where the reading distance can be sacrificed in favor of data capacity (e.g., security and authentication). Encoding of corporate documents, ballots, exams, etc., by directly printing the proposed tags on the item product to prevent counterfeiting is envisaged.

Expression and affinity purification of recombinant proteins from plants

NASA Technical Reports Server (NTRS)

Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

2002-01-01

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

Systems Biology of Recombinant Protein Production in Bacillus megaterium

NASA Astrophysics Data System (ADS)

Biedendieck, Rebekka; Bunk, Boyke; Fürch, Tobias; Franco-Lara, Ezequiel; Jahn, Martina; Jahn, Dieter

Over the last two decades the Gram-positive bacterium Bacillus megaterium was systematically developed to a useful alternative protein production host. Multiple vector systems for high yield intra- and extracellular protein production were constructed. Strong inducible promoters were combined with DNA sequences for optimised ribosome binding sites, various leader peptides for protein export and N- as well as C-terminal affinity tags for affinity chromatographic purification of the desired protein. High cell density cultivation and recombinant protein production were successfully tested. For further system biology based control and optimisation of the production process the genomes of two B. megaterium strains were completely elucidated, DNA arrays designed, proteome, fluxome and metabolome analyses performed and all data integrated using the bioinformatics platform MEGABAC. Now, solid theoretical and experimental bases for primary modeling attempts of the production process are available.

Interactome Analyses of Mature γ-Secretase Complexes Reveal Distinct Molecular Environments of Presenilin (PS) Paralogs and Preferential Binding of Signal Peptide Peptidase to PS2*

PubMed Central

Jeon, Amy Hye Won; Böhm, Christopher; Chen, Fusheng; Huo, Hairu; Ruan, Xueying; Ren, Carl He; Ho, Keith; Qamar, Seema; Mathews, Paul M.; Fraser, Paul E.; Mount, Howard T. J.; St George-Hyslop, Peter; Schmitt-Ulms, Gerold

2013-01-01

γ-Secretase plays a pivotal role in the production of neurotoxic amyloid β-peptides (Aβ) in Alzheimer disease (AD) and consists of a heterotetrameric core complex that includes the aspartyl intramembrane protease presenilin (PS). The human genome codes for two presenilin paralogs. To understand the causes for distinct phenotypes of PS paralog-deficient mice and elucidate whether PS mutations associated with early-onset AD affect the molecular environment of mature γ-secretase complexes, quantitative interactome comparisons were undertaken. Brains of mice engineered to express wild-type or mutant PS1, or HEK293 cells stably expressing PS paralogs with N-terminal tandem-affinity purification tags served as biological source materials. The analyses revealed novel interactions of the γ-secretase core complex with a molecular machinery that targets and fuses synaptic vesicles to cellular membranes and with the H+-transporting lysosomal ATPase macrocomplex but uncovered no differences in the interactomes of wild-type and mutant PS1. The catenin/cadherin network was almost exclusively found associated with PS1. Another intramembrane protease, signal peptide peptidase, predominantly co-purified with PS2-containing γ-secretase complexes and was observed to influence Aβ production. PMID:23589300

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

PubMed Central

Judge, Luke M.; Perez-Bermejo, Juan A.; Truong, Annie; Ribeiro, Alexandre J.S.; Yoo, Jennie C.; Jensen, Christina L.; Mandegar, Mohammad A.; Huebsch, Nathaniel; Kaake, Robyn M.; So, Po-Lin; Srivastava, Deepak; Krogan, Nevan J.

2017-01-01

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity. PMID:28724793

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

PubMed

Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R

2017-07-20

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli▿

PubMed Central

Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q. S.; Qiao, Chuanling

2007-01-01

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His6-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the Km and kcat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min−1, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling. PMID:17575004

Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative ProteomicsW⃞

PubMed Central

Majeran, Wojciech; Cai, Yang; Sun, Qi; van Wijk, Klaas J.

2005-01-01

Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway. PMID:16243905

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

PubMed

Castañón, Jesús; Román, José Pablo; Jessop, Theodore C; de Blas, Jesús; Haro, Rubén

2018-06-01

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

PubMed

Massiah, Michael A; Wright, Katharine M; Du, Haijuan

2016-04-01

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay. Copyright © 2016 John Wiley & Sons, Inc.

Recombinant antigens for immunodiagnosis of cystic echinococcosis

PubMed Central

Li, Jun; Zhang, Wen-Bao

2004-01-01

Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE) and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections. PMID:15188015

Toward biophysical probes for the 5-HT3 receptor: structure-activity relationship study of granisetron derivatives.

PubMed

Vernekar, Sanjeev Kumar V; Hallaq, Hasan Y; Clarkson, Guy; Thompson, Andrew J; Silvestri, Linda; Lummis, Sarah C R; Lochner, Martin

2010-03-11

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

Toward Biophysical Probes for the 5-HT3 Receptor: Structure−Activity Relationship Study of Granisetron Derivatives

PubMed Central

2010-01-01

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT3A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT3A receptors in mammalian cells. PMID:20146481

Targeting the UPR to Circumvent Endocrine Resistance in Breast Cancer

DTIC Science & Technology

2015-10-01

were submitted for the screen. Kinase assay protocol (DiscoverX): For most assays, kinase-tagged T7 phage strains were grown in parallel in 24-well...blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of...unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

PubMed

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

2013-12-01

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

Tags to Track Illicit Uranium and Plutonium

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

Haire, M. Jonathan; Forsberg, Charles W.

2007-07-01

With the expansion of nuclear power, it is essential to avoid nuclear materials from falling into the hands of rogue nations, terrorists, and other opportunists. This paper examines the idea of detection and attribution tags for nuclear materials. For a detection tag, it is proposed to add small amounts [about one part per billion (ppb)] of {sup 232}U to enriched uranium to brighten its radioactive signature. Enriched uranium would then be as detectable as plutonium and thus increase the likelihood of intercepting illicit enriched uranium. The use of rare earth oxide elements is proposed as a new type of 'attribution'more » tag for uranium and thorium from mills, uranium and plutonium fuels, and other nuclear materials. Rare earth oxides are chosen because they are chemically compatible with the fuel cycle, can survive high-temperature processing operations in fuel fabrication, and can be chosen to have minimal neutronic impact within the nuclear reactor core. The mixture of rare earths and/or rare earth isotopes provides a unique 'bar code' for each tag. If illicit nuclear materials are recovered, the attribution tag can identify the source and lot of nuclear material, and thus help police reduce the possible number of suspects in the diversion of nuclear materials based on who had access. (authors)« less

Off-Range Beaked Whale Studies (ORBS): Baseline Data and Tagging Development for Northern Bottlenose Whales (Hyperoodon ampulatus) off Jan Mayen, Norway

DTIC Science & Technology

2015-09-30

02.003’N, 07°01.981’W) To be recovered in 2016 Ranging code #08D1; releasing code #0803 In collaboration with Rune Hansen of the University of...the animal with PTT 134760 was tracked moving all the way south to the Azores Archipelago. Figure courtesy of Rune Hansen. Objective 4. conduct

Design of self-coded combinatorial libraries to facilitate direct analysis of ligands by mass spectrometry.

PubMed

Hughes, I

1998-09-24

The direct analysis of selected components from combinatorial libraries by sensitive methods such as mass spectrometry is potentially more efficient than deconvolution and tagging strategies since additional steps of resynthesis or introduction of molecular tags are avoided. A substituent selection procedure is described that eliminates the mass degeneracy commonly observed in libraries prepared by "split-and-mix" methods, without recourse to high-resolution mass measurements. A set of simple rules guides the choice of substituents such that all components of the library have unique nominal masses. Additional rules extend the scope by ensuring that characteristic isotopic mass patterns distinguish isobaric components. The method is applicable to libraries having from two to four varying substituent groups and can encode from a few hundred to several thousand components. No restrictions are imposed on the manner in which the "self-coded" library is synthesized or screened.

General Framework for Animal Food Safety Traceability Using GS1 and RFID

NASA Astrophysics Data System (ADS)

Cao, Weizhu; Zheng, Limin; Zhu, Hong; Wu, Ping

GS1 is global traceability standard, which is composed by the encoding system (EAN/UCC, EPC), the data carriers identified automatically (bar codes, RFID), electronic data interchange standards (EDI, XML). RFID is a non-contact, multi-objective automatic identification technique. Tracing of source food, standardization of RFID tags, sharing of dynamic data are problems to solve urgently for recent traceability systems. The paper designed general framework for animal food safety traceability using GS1 and RFID. This framework uses RFID tags encoding with EPCglobal tag data standards. Each information server has access tier, business tier and resource tier. These servers are heterogeneous and distributed, providing user access interfaces by SOAP or HTTP protocols. For sharing dynamic data, discovery service and object name service are used to locate dynamic distributed information servers.

Use of the QR Reader to Provide Real-Time Evaluation of Residents' Skills Following Surgical Procedures.

PubMed

Reynolds, Kellin; Barnhill, Danny; Sias, Jamie; Young, Amy; Polite, Florencia Greer

2014-12-01

A portable electronic method of providing instructional feedback and recording an evaluation of resident competency immediately following surgical procedures has not previously been documented in obstetrics and gynecology. This report presents a unique electronic format that documents resident competency and encourages verbal communication between faculty and residents immediately following operative procedures. The Microsoft Tag system and SurveyMonkey platform were linked by a 2-D QR code using Microsoft QR code generator. Each resident was given a unique code (TAG) embedded onto an ID card. An evaluation form was attached to each resident's file in SurveyMonkey. Postoperatively, supervising faculty scanned the resident's TAG with a smartphone and completed the brief evaluation using the phone's screen. The evaluation was reviewed with the resident and automatically submitted to the resident's educational file. The evaluation system was quickly accepted by residents and faculty. Of 43 residents and faculty in the study, 38 (88%) responded to a survey 8 weeks after institution of the electronic evaluation system. Thirty (79%) of the 38 indicated it was superior to the previously used handwritten format. The electronic system demonstrated improved utilization compared with paper evaluations, with a mean of 23 electronic evaluations submitted per resident during a 6-month period versus 14 paper assessments per resident during an earlier period of 6 months. This streamlined portable electronic evaluation is an effective tool for direct, formative feedback for residents, and it creates a longitudinal record of resident progress. Satisfaction with, and use of, this evaluation system was high.

Use of the QR Reader to Provide Real-Time Evaluation of Residents' Skills Following Surgical Procedures

PubMed Central

Reynolds, Kellin; Barnhill, Danny; Sias, Jamie; Young, Amy; Polite, Florencia Greer

2014-01-01

Background A portable electronic method of providing instructional feedback and recording an evaluation of resident competency immediately following surgical procedures has not previously been documented in obstetrics and gynecology. Objective This report presents a unique electronic format that documents resident competency and encourages verbal communication between faculty and residents immediately following operative procedures. Methods The Microsoft Tag system and SurveyMonkey platform were linked by a 2-D QR code using Microsoft QR code generator. Each resident was given a unique code (TAG) embedded onto an ID card. An evaluation form was attached to each resident's file in SurveyMonkey. Postoperatively, supervising faculty scanned the resident's TAG with a smartphone and completed the brief evaluation using the phone's screen. The evaluation was reviewed with the resident and automatically submitted to the resident's educational file. Results The evaluation system was quickly accepted by residents and faculty. Of 43 residents and faculty in the study, 38 (88%) responded to a survey 8 weeks after institution of the electronic evaluation system. Thirty (79%) of the 38 indicated it was superior to the previously used handwritten format. The electronic system demonstrated improved utilization compared with paper evaluations, with a mean of 23 electronic evaluations submitted per resident during a 6-month period versus 14 paper assessments per resident during an earlier period of 6 months. Conclusions This streamlined portable electronic evaluation is an effective tool for direct, formative feedback for residents, and it creates a longitudinal record of resident progress. Satisfaction with, and use of, this evaluation system was high. PMID:26140128

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

Rowe, Nathan C; Younkin, James R; Pickett, Chris A

Radio-Frequency (RF) devices have revolutionized many aspects of modern industrial processes. RF technology can enable wireless communication for tag identification, sensor communication, and asset tracking. Radio-frequency identification (RFID) is a technology that utilizes wireless communication to interrogate and identify an electronic tag attached to an item in order to identify the item. The technology can come in many forms: passive or active tags, low to ultra-wideband frequencies, small paper-thin tags to brick-sized units, and simple tags or highly integrated sensor packages. RF technology, and specifically RFID, has been applied widely in commercial markets for inventory, supply chain management, and assetmore » tracking. Several recent studies have demonstrated the safeguards benefits of utilizing RFID versus conventional inventory tagging methods for tracking nuclear material. These studies have indicated that the RF requirements for safeguards functions are more stringent than the RF requirements for other inventory tracking and accounting applications. Additionally, other requirements must be addressed, including environmental and operating conditions, authentication, and tag location and attachment. Facility restrictions on radio spectrum, method of tag attachment, and sensitivity of the data collected impact the tag selection and system design. More important, the intended use of the system must be considered. The requirements for using RF to simply replace or supplement container identifiers such as bar codes that facilitate the inventory function will differ greatly from the requirements for deploying RF for unattended monitoring applications. Several studies have investigated these considerations to advance commercial RF devices for safeguards use, and a number of system concepts have been developed. This paper will provide an overview of past studies and current technologies, and will investigate the requirements, existing gaps, and several potential next steps for advancing RF techniques for safeguards use.« less

How the brain assigns a neural tag to arbitrary points in a high-dimensional space

NASA Astrophysics Data System (ADS)

Stevens, Charles

Brains in almost all organisms need to deal with very complex stimuli. For example, most mammals are very good at face recognition, and faces are very complex objects indeed. For example, modern face recognition software represents a face as a point in a 10,000 dimensional space. Every human must be able to learn to recognize any of the 7 billion faces in the world, and can recognize familiar faces after a display of the face is viewed for only a few hundred milliseconds. Because we do not understand how faces are assigned locations in a high-dimensional space by the brain, attacking the problem of how face recognition is accomplished is very difficult. But a much easier problem of the same sort can be studied for odor recognition. For the mouse, each odor is assigned a point in a 1000 dimensional space, and the fruit fly assigns any odor a location in only a 50 dimensional space. A fly has about 50 distinct types of odorant receptor neurons (ORNs), each of which produce nerve impulses at a specific rate for each different odor. This pattern of firing produced across 50 ORNs is called `a combinatorial odor code', and this code assigns every odor a point in a 50 dimensional space that is used to identify the odor. In order to learn the odor, the brain must alter the strength of synapses. The combinatorial code cannot itself by used to change synaptic strength because all odors use same neurons to form the code, and so all synapses would be changed for any odor and the odors could not be distinguished. In order to learn an odor, the brain must assign a set of neurons - the odor tag - that have the property that these neurons (1) should make use of all of the information available about the odor, and (2) insure that any two tags overlap as little as possible (so one odor does not modify synapses used by other odors). In the talk, I will explain how the olfactory system of both the fruit fly and the mouse produce a tag for each odor that has these two properties. Supported by NSF.

Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3)

PubMed Central

Austin, Brian P.; Waugh, David S.

2011-01-01

The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25 mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5 mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4 °C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration. PMID:22197595

Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification.

PubMed

Liu, Hu; Dong, Xiaoyan; Sun, Yan

2016-01-15

A series of highly charged nanoscale chelators were fabricated by grafting of poly(glycidyl methacrylate-iminodiacetic acid) (pGI) chains with iminodiacetic acid (IDA) chelating group on silica nanoparticles (SNPs) via atom transfer radical polymerization (ATRP). The nanoscale chelators, denoted as SNPs-pGI, possessed a nickel ion chelating capacity as high as 2800 μmol/g, 50 times higher than the IDA-modified Sepharose FF (IDA-Sepharose) resin reported in literature and offered a high affinity binding capacity for hexahistidine-tagged enhanced green fluorescence protein (6 × His-EGFP) after nickel ion loading. More importantly, the anionic SNPs-pGI of high charge densities displayed much better performance than IDA-Sepharose in facilitating the refolding of like-charged 6 × His-EGFP from inclusion bodies (IBs). For example, for 0.2mg/mL 6 × His-EGFP IB refolding, addition of 6.2 μL/mL SNPs-pGI with the highest charge density led to a refolding yield of 90%, over 43% higher than that obtained with 460 μL/mL IDA-Sepharose. It is notable that the much higher efficiency of the nanoscale chelator was obtained with a chelator consumption corresponding to only 1.4% of IDA-Sepharose. Moreover, the highly charged SNPs-pGI could efficiently facilitate the refolding of 6 × His-EGFP at higher IB concentrations (0.4 and 0.8 mg/mL). After refolding, nickel ions addition led to the recovery of the refolded 6 × His-EGFP with high yield (80%), purity (96%) and enrichment ratio (1.8). All the results suggest that the SNPs-pGI of high charge densities were promising for cost-effective recovery of His-tagged proteins expressed as IBs with the integrative like-charge facilitated refolding and metal-chelate affinity purification strategy. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Hervey, IV, William Judson; Khalsa-Moyers, Gurusahai K; Lankford, Patricia K

Protein enrichments of engineered, affinity-tagged (or bait ) fusion proteins with interaction partners are often laden with background, non-specific proteins, due to interactions that occur in vitro as an artifact of the technique. Furthermore, the in vivo expression of the bait protein may itself affect physiology or metabolism. In this study, intrinsic affinity purification challenges were investigated in a model protein complex, DNA-dependent RNA polymerase (RNAP), encompassing chromosome- and plasmid-encoding strategies for bait proteins in two different microbial species: Escherichia coli and Rhodopseudomonas palustris. Isotope ratio measurements of bait protein expression strains relative to native, wild-type strains were performed bymore » liquid chromatography tandem mass spectrometry (LC-MS-MS) to assess bait protein expression strategies in each species. Authentic interacting proteins of RNAP were successfully discerned from artifactual co-isolating proteins by the isotopic differentiation of interactions as random or targeted (I-DIRT) method (A. J. Tackett et al. J. Proteome Res. 2005, 4 (5), 1752-1756). To investigate broader effects of bait protein production in the bacteria, we compared proteomes from strains harboring a plasmid that encodes an affinity-tagged subunit (RpoA) of the RNAP complex with the corresponding wild-type strains using stable isotope metabolic labeling. The ratio of RpoA abundance in plasmid strains versus wild type was 0.8 for R. palustris and 1.7 for E. coli. While most other proteins showed no appreciable difference, proteins significantly increased in abundance in plasmid-encoded bait-expressing strains of both species included the plasmid encoded antibiotic resistance protein, GenR and proteins involved in amino acid biosynthesis. Together, these local, complex-specific and more global, whole proteome isotopic abundance ratio measurements provided a tool for evaluating both in vivo and in vitro effects of plasmid-encoding strategies for bait protein expression. This approach has the potential for enabling discovery of protein-protein interactions among the growing number of sequenced microbial species without the need for development of chromosomal insertion systems.« less

Glycan reductive isotope labeling for quantitative glycomics.

PubMed

Xia, Baoyun; Feasley, Christa L; Sachdev, Goverdhan P; Smith, David F; Cummings, Richard D

2009-04-15

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [(12)C(6)]aniline and [(13)C(6)]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.

GLYCAN REDUCTIVE ISOTOPE LABELING (GRIL) FOR QUANTITATIVE GLYCOMICS

PubMed Central

Xia, Baoyun; Feasley, Christa L.; Sachdev, Goverdhan P.; Smith, David F.; Cummings, Richard D.

2009-01-01

Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed Glycan Reductive Isotope Labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [12C6]-aniline and [13C6]-aniline. These dual-labeled aniline-tagged glycans can be recovered by reversed-phase chromatography and quantified based on UV-absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins using this method. This technique allows for linear, relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of Glycomics. PMID:19454239

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

PubMed Central

Campos, Magnólia de A; Silva, Marilia S; Magalhães, Cláudio P; Ribeiro, Simone G; Sarto, Rafael PD; Vieira, Eduardo A; Grossi de Sá, Maria F

2008-01-01

Background Heterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity. Results To validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His6-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni2+) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions. Conclusion Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications. PMID:18334031

Functional efficacy of human recombinant FGF-2s tagged with (His)6 and (His-Asn)6 at the N- and C-termini in human gingival fibroblast and periodontal ligament-derived cells.

PubMed

Lee, Ji-Hye; Lee, Ji-Eun; Kang, Kyung-Jung; Jang, Young-Joo

2017-07-01

Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H 6 ) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN 6 ) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 ) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H 6 -FGF2, FGF2-H 6 , HN 6 -FGF2, and FGF2-HN 6 were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme.

PubMed

Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

2017-10-01

Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

Novel 6xHis tagged foot-and-mouth disease virus vaccine bound to nanolipoprotein adjuvant via metal ions provides antigenic distinction and effective protective immunity

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

Rai, Devendra K.; Segundo, Fayna Diaz-San; Department of Pathobiology and Veterinary Science, CANR, University of Connecticut, Storrs, CT 06269

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co{sup 2+} affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni{sup 2+}-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDVmore » neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance. - Highlights: • 6xHis-tags in A{sub 24} FMDV enable purification and biding to adjuvants via metal ions. • 6xHis A{sub 24} FMDV:MPLA:NiNLP vaccine enhanced protective immunity against FMDV. • Surface exposed capsid tags allow distinction of infected from vaccinated animals.« less

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

PubMed

Boeneman, Kelly; Fossum, Solveig; Yang, Yanhua; Fingland, Nicholas; Skarstad, Kirsten; Crooke, Elliott

2009-05-01

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins. The library was screened for the ability to rescue dnaA(ts) mutants, and a candidate gfp-dnaA was used to replace the dnaA gene of wild-type cells. The resulting cells produce close to physiological levels of GFP-DnaA from the endogenous promoter as their only source of DnaA and somewhat under-initiate replication with moderate asynchrony. Visualization of GFP-tagged DnaA in living cells revealed that DnaA adopts a helical pattern that spirals along the long axis of the cell, a pattern also seen in wild-type cells by immunofluorescence with affinity purified anti-DnaA antibody. Although the DnaA helices closely resemble the helices of the actin analogue MreB, co-visualization of GFP-tagged DnaA and RFP-tagged MreB demonstrates that DnaA and MreB adopt discrete helical structures along the length of the longitudinal cell axis.

Thermal ecology of subadult and adult muskellunge in a thermally enriched reservoir

USGS Publications Warehouse

Cole, A. J.; Bettoli, Phillip William

2014-01-01

The movement of adult muskellunge, Esox masquinongy Mitchill, has been investigated in a variety of systems, but temperature selection by muskellunge has not been examined where well-oxygenated waters were available over a range of temperatures for much of the year. Thirty subadult and adult muskellunge tagged internally with temperature-sensing radio tags were tracked from March 2010 to March 2011 in a Tennessee reservoir. Mean tag temperatures were 18.9 °C in spring (March to May), 22.1 °C in summer (June to August), 16.5 °C in autumn and 9.8 °C in winter (December to February). When the greatest range in water temperatures was available (7.1–33.3 °C; May to early August 2010), their realised thermal niche (mean ± 1 SD) was 22.3 °C ± 1.8; the realised thermal niche was affected by fish size (smaller fish selected slightly warmer temperatures) but not sex. An electric generating steam plant discharging warm water resumed operation in January 2011, and most (86%) tagged fish occupied the plume where temperatures were ≈10 °C warmer than ambient water temperatures. No mortalities were observed 15 days later when plant operations ceased. Their affinity for the heated plume prompted concerns that muskellunge will be too easily exploited when the plant operates during winter.

Structural characterization of core-bradavidin in complex with biotin

PubMed Central

Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

2017-01-01

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

A recombinant estrogen receptor fragment-based homogeneous fluorescent assay for rapid detection of estrogens.

PubMed

Wang, Dan; Xie, Jiangbi; Zhu, Xiaocui; Li, Jinqiu; Zhao, Dongqin; Zhao, Meiping

2014-05-15

In this work, we demonstrate a novel estrogenic receptor fragment-based homogeneous fluorescent assay which enables rapid and sensitive detection of 17β-estradiol (E2) and other highly potent estrogens. A modified human estrogenic receptor fragment (N-His × 6-hER270-595-C-Strep tag II) has been constructed that contains amino acids 270-595 of wild-type human estrogenic receptor α (hER270-595) and two specific tags (6 × His and Strep tag II) fused to the N and C terminus, respectively. The designed receptor protein fragment could be easily produced by prokaryotic expression with high yield and high purity. The obtained protein exhibits high binding affinity to E2 and the two tags greatly facilitate the application of the recombinant protein. Taking advantage of the unique spectroscopic properties of coumestrol (CS), a fluorescent phytoestrogen, a CS/hER270-595-based fluorescent assay has been developed which can sensitively respond to E2 within 1.0 min with a linear working range from 0.1 to 20 ng/mL and a limit of detection of 0.1 ng/mL. The assay was successfully applied for rapid detection of E2 in the culture medium of rat hippocampal neurons. The method also holds great potential for high-throughput monitoring the variation of estrogen levels in complex biological fluids, which is crucial for investigation of the molecular basis of various estrogen-involved processes. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.

PubMed

Chen, Yong; Li, Yang; Liu, Peng; Sun, Qun; Liu, Zhu

2014-04-01

A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant.

PubMed

Alves, Nathan J; Turner, Kendrick B; DiVito, Kyle A; Daniele, Michael A; Walper, Scott A

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function. Published by Elsevier Masson SAS.

Security Enhancement Mechanism Based on Contextual Authentication and Role Analysis for 2G-RFID Systems

PubMed Central

Tang, Wan; Chen, Min; Ni, Jin; Yang, Ximin

2011-01-01

The traditional Radio Frequency Identification (RFID) system, in which the information maintained in tags is passive and static, has no intelligent decision-making ability to suit application and environment dynamics. The Second-Generation RFID (2G-RFID) system, referred as 2G-RFID-sys, is an evolution of the traditional RFID system to ensure better quality of service in future networks. Due to the openness of the active mobile codes in the 2G-RFID system, the realization of conveying intelligence brings a critical issue: how can we make sure the backend system will interpret and execute mobile codes in the right way without misuse so as to avoid malicious attacks? To address this issue, this paper expands the concept of Role-Based Access Control (RBAC) by introducing context-aware computing, and then designs a secure middleware for backend systems, named Two-Level Security Enhancement Mechanism or 2L-SEM, in order to ensure the usability and validity of the mobile code through contextual authentication and role analysis. According to the given contextual restrictions, 2L-SEM can filtrate the illegal and invalid mobile codes contained in tags. Finally, a reference architecture and its typical application are given to illustrate the implementation of 2L-SEM in a 2G-RFID system, along with the simulation results to evaluate how the proposed mechanism can guarantee secure execution of mobile codes for the system. PMID:22163983

Security enhancement mechanism based on contextual authentication and role analysis for 2G-RFID systems.

PubMed

Tang, Wan; Chen, Min; Ni, Jin; Yang, Ximin

2011-01-01

The traditional Radio Frequency Identification (RFID) system, in which the information maintained in tags is passive and static, has no intelligent decision-making ability to suit application and environment dynamics. The Second-Generation RFID (2G-RFID) system, referred as 2G-RFID-sys, is an evolution of the traditional RFID system to ensure better quality of service in future networks. Due to the openness of the active mobile codes in the 2G-RFID system, the realization of conveying intelligence brings a critical issue: how can we make sure the backend system will interpret and execute mobile codes in the right way without misuse so as to avoid malicious attacks? To address this issue, this paper expands the concept of Role-Based Access Control (RBAC) by introducing context-aware computing, and then designs a secure middleware for backend systems, named Two-Level Security Enhancement Mechanism or 2L-SEM, in order to ensure the usability and validity of the mobile code through contextual authentication and role analysis. According to the given contextual restrictions, 2L-SEM can filtrate the illegal and invalid mobile codes contained in tags. Finally, a reference architecture and its typical application are given to illustrate the implementation of 2L-SEM in a 2G-RFID system, along with the simulation results to evaluate how the proposed mechanism can guarantee secure execution of mobile codes for the system.

ELROI Extremely Low Resource Optical Identifier. A license plate for your satellite, and more.

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

Palmer, David

ELROI (Extremely Low Resource Optical Identifier) is a license plate for your satellite; a small tag that flashes an optical identification code that can be read by a small telescope on the ground. The final version of the tag will be the size of a thick postage stamp and fully autonomous: you can attach it to everything that goes into space, including small cubesats and inert debris like rocket stages, and it will keep blinking even after the satellite is shut down, reliably identifying the object from launch until re-entry.

Tracking animals in freshwater with electronic tags: past, present and future

USGS Publications Warehouse

Cooke, Steven J.; Midwood, Jonathan D.; Thiem, Jason D.; Klimley, Peter; Lucas, Martyn C.; Thorstad, Eva B.; Eiler, John; Holbrook, Chris; Ebner, Brendan C.

2013-01-01

Considerable technical developments over the past half century have enabled widespread application of electronic tags to the study of animals in the wild, including in freshwater environments. We review the constraints associated with freshwater telemetry and biologging and the technical developments relevant to their use. Technical constraints for tracking animals are often influenced by the characteristics of the animals being studied and the environment they inhabit. Collectively, they influence which and how technologies can be used and their relative effectiveness. Although radio telemetry has historically been the most commonly used technology in freshwater, passive integrated transponder (PIT) technology, acoustic telemetry and biologgers are becoming more popular. Most telemetry studies have focused on fish, although an increasing number have focused on other taxa, such as turtles, crustaceans and molluscs. Key technical developments for freshwater systems include: miniaturization of tags for tracking small-size life stages and species, fixed stations and coded tags for tracking large samples of animals over long distances and large temporal scales, inexpensive PIT systems that enable mass tagging to yield population- and community-level relevant sample sizes, incorporation of sensors into electronic tags, validation of tag attachment procedures with a focus on maintaining animal welfare, incorporation of different techniques (for example, genetics, stable isotopes) and peripheral technologies (for example, geographic information systems, hydroacoustics), development of novel analytical techniques, and extensive international collaboration. Innovations are still needed in tag miniaturization, data analysis and visualization, and in tracking animals over larger spatial scales (for example, pelagic areas of lakes) and in challenging environments (for example, large dynamic floodplain systems, under ice). There seems to be a particular need for adapting various global positioning system and satellite tagging approaches to freshwater. Electronic tagging provides a mechanism to collect detailed information from imperilled animals and species that have no direct economic value. Current and future advances will continue to improve our knowledge of the natural history of aquatic animals and ecological processes in freshwater ecosystems while facilitating evidence-based resource management and conservation.

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

Security authentication with a three-dimensional optical phase code using random forest classifier: an overview

NASA Astrophysics Data System (ADS)

Markman, Adam; Carnicer, Artur; Javidi, Bahram

2017-05-01

We overview our recent work [1] on utilizing three-dimensional (3D) optical phase codes for object authentication using the random forest classifier. A simple 3D optical phase code (OPC) is generated by combining multiple diffusers and glass slides. This tag is then placed on a quick-response (QR) code, which is a barcode capable of storing information and can be scanned under non-uniform illumination conditions, rotation, and slight degradation. A coherent light source illuminates the OPC and the transmitted light is captured by a CCD to record the unique signature. Feature extraction on the signature is performed and inputted into a pre-trained random-forest classifier for authentication.

Digital barcodes of suspension array using laser induced breakdown spectroscopy

PubMed Central

He, Qinghua; Liu, Yixi; He, Yonghong; Zhu, Liang; Zhang, Yilong; Shen, Zhiyuan

2016-01-01

We show a coding method of suspension array based on the laser induced breakdown spectroscopy (LIBS), which promotes the barcodes from analog to digital. As the foundation of digital optical barcodes, nanocrystals encoded microspheres are prepared with self-assembly encapsulation method. We confirm that digital multiplexing of LIBS-based coding method becomes feasible since the microsphere can be coded with direct read-out data of wavelengths, and the method can avoid fluorescence signal crosstalk between barcodes and analyte tags, which lead to overall advantages in accuracy and stability to current fluorescent multicolor coding method. This demonstration increases the capability of multiplexed detection and accurate filtrating, expanding more extensive applications of suspension array in life science. PMID:27808270

48 CFR 252.211-7006 - Passive Radio Frequency Identification.

Code of Federal Regulations, 2013 CFR

2013-10-01

... radio frequency identification (RFID) or item unique identification (IUID) information, order... CodeTM (EPC®) means an identification scheme for universally identifying physical objects via RFID tags... passive RFID technology. Exterior container means a MIL-STD-129 defined container, bundle, or assembly...

48 CFR 252.211-7006 - Passive Radio Frequency Identification.

Code of Federal Regulations, 2012 CFR

2012-10-01

... radio frequency identification (RFID) or item unique identification (IUID) information, order... CodeTM (EPC®) means an identification scheme for universally identifying physical objects via RFID tags... passive RFID technology. Exterior container means a MIL-STD-129 defined container, bundle, or assembly...

48 CFR 252.211-7006 - Passive Radio Frequency Identification.

Code of Federal Regulations, 2014 CFR

2014-10-01

... radio frequency identification (RFID) or item unique identification (IUID) information, order... CodeTM (EPC®) means an identification scheme for universally identifying physical objects via RFID tags... passive RFID technology. Exterior container means a MIL-STD-129 defined container, bundle, or assembly...

Heterologous expression of a thermophilic diacylglycerol acyltransferase triggers triglyceride accumulation in Escherichia coli.

PubMed

Lázaro, Beatriz; Villa, Juan A; Santín, Omar; Cabezas, Matilde; Milagre, Cintia D F; de la Cruz, Fernando; Moncalián, Gabriel

2017-01-01

Triglycerides (TAGs), the major storage molecules of metabolic energy and source of fatty acids, are produced as single cell oil by some oleogenic microorganisms. However, these microorganisms require strict culture conditions, show low carbon source flexibilities, lack efficient genetic modification tools and in some cases pose safety concerns. TAGs have essential applications such as behaving as a source for added-value fatty acids or giving rise to the production of biodiesel. Hence, new alternative methods are urgently required for obtaining these oils. In this work we describe TAG accumulation in the industrially appropriate microorganism Escherichia coli expressing the heterologous enzyme tDGAT, a wax ester synthase/triacylglycerol:acylCoA acyltranferase (WS/DGAT). With this purpose, we introduce a codon-optimized gene from the thermophilic actinomycete Thermomonospora curvata coding for a WS/DGAT into different E. coli strains, describe the metabolic effects associated to the expression of this protein and evaluate neutral lipid accumulation. We observe a direct relation between the expression of this WS/DGAT and TAG production within a wide range of culture conditions. More than 30% TAGs were detected within the bacterial neutral lipids in 90 minutes after induction. TAGs were observed to be associated with the hydrophobic enzyme while forming round intracytoplasmic bodies, which could represent a bottleneck for lipid accumulation in E. coli. We detected an increase of almost 3-fold in the monounsaturated fatty acids (MUFA) occurring in the recombinant strains. These MUFA were predominant in the accumulated TAGs achieving 46% of the TAG fatty acids. These results set the basis for further research on the achievement of a suitable method towards the sustainable production of these neutral lipids.

Bicarbonate Increases Binding Affinity of Vibrio cholerae ToxT to Virulence Gene Promoters

PubMed Central

Thomson, Joshua J.

2014-01-01

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription. PMID:25182489

Integrated amperometric affinity biosensors using Co2+-tetradentate nitrilotriacetic acid modified disposable carbon electrodes: application to the determination of β-lactam antibiotics.

PubMed

Conzuelo, Felipe; Gamella, María; Campuzano, Susana; Martínez-Ruiz, Paloma; Esteban-Torres, María; de las Rivas, Blanca; Reviejo, A Julio; Muñoz, Rosario; Pingarrón, José M

2013-03-19

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co(2+)-tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at -0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min.

Advances in identification and validation of protein targets of natural products without chemical modification.

PubMed

Chang, J; Kim, Y; Kwon, H J

2016-05-04

Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

PubMed

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-03-22

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

PubMed Central

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-01-01

A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

The Dose–Response Relationship Between Tobacco Education Advertising and Calls to Quitlines in the United States, March–June, 2012

PubMed Central

Alexander, Robert L.; Shafer, Paul; Mann, Nathan; Malarcher, Ann; Zhang, Lei

2015-01-01

Introduction We estimated changes in call volume in the United States in response to increases in advertising doses of the Tips From Former Smokers (Tips) campaign, the first federal national tobacco education campaign, which aired for 12 weeks from March 19 to June 10, 2012. We also measured the effectiveness of ad taglines that promoted calls directly with a quitline number (1-800-QUIT-NOW) and indirectly with a cessation help website (Smokefree.gov). Methods Multivariate regressions estimated the weekly number of calls to 1–800-QUIT-NOW by area code as a function of weekly market-level gross rating points (GRPs) from CDC’s Tips campaign in 2012. The number of quitline calls attributable solely to Tips was predicted. Results For quitline-tagged ads, an additional 100 television GRPs per week was associated with an increase of 89 calls per week in a typical area code in the United States (P < .001). The same unit increase in advertising GRPs for ads tagged with Smokefree.gov was associated with an increase of 29 calls per week in any given area code (P < .001). We estimated that the Tips campaign was responsible for more than 170,000 additional calls to 1–800-QUIT-NOW during the campaign and that it would have generated approximately 140,000 additional calls if all ads were tagged with 1–800-QUIT-NOW. Conclusion For campaign planners, these results make it possible to estimate 1) the likely impact of tobacco prevention media buys and 2) the additional quitline capacity needed at the national level should future campaigns of similar scale use 1–800-QUIT-NOW taglines exclusively. PMID:26542143

The Dose-Response Relationship Between Tobacco Education Advertising and Calls to Quitlines in the United States, March-June, 2012.

PubMed

Davis, Kevin C; Alexander, Robert L; Shafer, Paul; Mann, Nathan; Malarcher, Ann; Zhang, Lei

2015-11-05

We estimated changes in call volume in the United States in response to increases in advertising doses of the Tips From Former Smokers (Tips) campaign, the first federal national tobacco education campaign, which aired for 12 weeks from March 19 to June 10, 2012. We also measured the effectiveness of ad taglines that promoted calls directly with a quitline number (1-800-QUIT-NOW) and indirectly with a cessation help website (Smokefree.gov). Multivariate regressions estimated the weekly number of calls to 1-800-QUIT-NOW by area code as a function of weekly market-level gross rating points (GRPs) from CDC's Tips campaign in 2012. The number of quitline calls attributable solely to Tips was predicted. For quitline-tagged ads, an additional 100 television GRPs per week was associated with an increase of 89 calls per week in a typical area code in the United States (P < .001). The same unit increase in advertising GRPs for ads tagged with Smokefree.gov was associated with an increase of 29 calls per week in any given area code (P < .001). We estimated that the Tips campaign was responsible for more than 170,000 additional calls to 1-800-QUIT-NOW during the campaign and that it would have generated approximately 140,000 additional calls if all ads were tagged with 1-800-QUIT-NOW. For campaign planners, these results make it possible to estimate 1) the likely impact of tobacco prevention media buys and 2) the additional quitline capacity needed at the national level should future campaigns of similar scale use 1-800-QUIT-NOW taglines exclusively.

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

Simultaneously achieve soluble expression and biomimetic immobilization of Candida antarctica lipase B by introducing polyamine tags.

PubMed

Zhou, Xiaoxue; Han, Yu; Lv, Zheng; Tian, Xuemei; Li, Han; Xie, Panpan; Zheng, Liangyu

2017-05-10

Polyamine tags fused in Candida antarctica lipase B (CalB) can help achieve high soluble expression of CalB in E. coli and can directly mediate silicification, which leads to rapid formation of a CalB-silica particle complex through a one-step approach. After optimization experiments, the fused lipase CalB tagged with 6-histidine at the N terminal and 10-lysine at the C terminal (6His-CalB-10Lys) is effectively expressed with high solubility (0.1mg/mL) and specific activity (10.1U/mg), and easily cross-linked in silica particles with a high immobilization efficiency of 96.8% and activity recovery of 81.5%. The immobilized lipase 6His-CalB-10Lys exhibits excellent performance at broad temperature ranges, high thermal and storage stabilities, and superior reusability. Michaelis-Menten kinetics indicates that the affinity and enantioselectivity of the free and immobilized 6His-CalB-10Lys toward the substrate are better than that of commercial Novozym 435 in enantioselective resolution of (S)-N-(2-ethyl-6-methylphenyl) alanine ((S)-NEMPA). The strategies described in this paper are useful for the facile expression and construction of diverse enzyme systems with high efficiency and excellent recyclability. Copyright © 2017 Elsevier B.V. All rights reserved.

Dual-Valve and Counter-Flow Surface Plasmon Resonance.

PubMed

Wang, Xiaoying; Zhou, Feimeng

2018-04-17

Two six-port injector valves and one selector valve commonly used in flow injection analysis are combined with a surface plasmon resonance (SPR) instrument wherein solutions introduced from the two inlets counter-flow inside the flow cell. The system is versatile as the same or different solutions can be rapidly and repeatedly introduced to the two fluidic channels in series or in parallel. Unlike most commercial SPR instruments employing a single injector valve, solutions separately injected from the two injector valves can be readily exchanged (<1 s) between the two channels. This new method, referred to as the alternate injection mode, not only saves analysis time but also facilitates efficient and facile surface reactions for ligand immobilization and prevents immobilized species from desorbing. These advantages are demonstrated with the measurements of binding of acetazolamide (222.2 Da) to histidine-tagged human carbonic anhydrase II (his-tagged HCA). Amine-containing residues of his-tagged HCA molecules tethered at Ni-nitrilotriacetic acid (NTA) sensors were rapidly cross-linked to the underlying carboxymethylated dextran. The higher ligand densities and more stable surfaces are essential for SPR detection of small molecule binding. In a different application, microglobulin solutions of increasing concentrations were introduced for continuous binding to the preimmobilized antibody. The kinetic and affinity measurements can be conducted without performing repeated dissociation and surface regeneration reactions.

Comparing the Properties of Electrochemical-Based DNA Sensors Employing Different Redox Tags

PubMed Central

Kang, Di; Zuo, Xiaolei; Yang, Renqiang; Xia, Fan; Plaxco, Kevin W.; White, Ryan J.

2009-01-01

Many electrochemical biosensor approaches developed in recent years utilize redox labeled (most commonly methylene blue or ferrocene) oligonucleotide probes site-specifically attached to an interrogating electrode. Sensors in this class have been reported employing a range of probe architectures, including single- and double-stranded DNA, more complex DNA structures, DNA and RNA aptamers and, most recently, DNA-small molecule chimeras. Signaling in this class of sensors is generally predicated on binding-induced changes in the efficiency with which the covalently attached redox label transfers electrons with the interrogating electrode. Here we have investigated how the properties of the redox tag affect the performance of such sensors. Specifically, we compare the differences in signaling and stability of electrochemical DNA sensors (E-DNA sensors) fabricated using either ferrocene or methylene blue as the signaling redox moiety. We find that while both tags support efficient E-DNA signaling, ferrocene produces slightly improved signal gain and target affinity. These small advantages, however, come at a potentially significant price: the ferrocene-based sensors are far less stable than their methylene blue counterparts, particularly with regards to stability to long-term storage, repeated electrochemical interrogations, repeated sensing/regeneration iterations, and employment in complex sample matrices such as blood serum. PMID:19810694

On origin of genetic code and tRNA before translation

PubMed Central

2011-01-01

Background Synthesis of proteins is based on the genetic code - a nearly universal assignment of codons to amino acids (aas). A major challenge to the understanding of the origins of this assignment is the archetypal "key-lock vs. frozen accident" dilemma. Here we re-examine this dilemma in light of 1) the fundamental veto on "foresight evolution", 2) modular structures of tRNAs and aminoacyl-tRNA synthetases, and 3) the updated library of aa-binding sites in RNA aptamers successfully selected in vitro for eight amino acids. Results The aa-binding sites of arginine, isoleucine and tyrosine contain both their cognate triplets, anticodons and codons. We have noticed that these cases might be associated with palindrome-dinucleotides. For example, one-base shift to the left brings arginine codons CGN, with CG at 1-2 positions, to the respective anticodons NCG, with CG at 2-3 positions. Formally, the concomitant presence of codons and anticodons is also expected in the reverse situation, with codons containing palindrome-dinucleotides at their 2-3 positions, and anticodons exhibiting them at 1-2 positions. A closer analysis reveals that, surprisingly, RNA binding sites for Arg, Ile and Tyr "prefer" (exactly as in the actual genetic code) the anticodon(2-3)/codon(1-2) tetramers to their anticodon(1-2)/codon(2-3) counterparts, despite the seemingly perfect symmetry of the latter. However, since in vitro selection of aa-specific RNA aptamers apparently had nothing to do with translation, this striking preference provides a new strong support to the notion of the genetic code emerging before translation, in response to catalytic (and possibly other) needs of ancient RNA life. Consistently with the pre-translation origin of the code, we propose here a new model of tRNA origin by the gradual, Fibonacci process-like, elongation of a tRNA molecule from a primordial coding triplet and 5'DCCA3' quadruplet (D is a base-determinator) to the eventual 76 base-long cloverleaf-shaped molecule. Conclusion Taken together, our findings necessarily imply that primordial tRNAs, tRNA aminoacylating ribozymes, and (later) the translation machinery in general have been co-evolving to ''fit'' the (likely already defined) genetic code, rather than the opposite way around. Coding triplets in this primal pre-translational code were likely similar to the anticodons, with second and third nucleotides being more important than the less specific first one. Later, when the code was expanding in co-evolution with the translation apparatus, the importance of 2-3 nucleotides of coding triplets "transferred" to the 1-2 nucleotides of their complements, thus distinguishing anticodons from codons. This evolutionary primacy of anticodons in genetic coding makes the hypothesis of primal stereo-chemical affinity between amino acids and cognate triplets, the hypothesis of coding coenzyme handles for amino acids, the hypothesis of tRNA-like genomic 3' tags suggesting that tRNAs originated in replication, and the hypothesis of ancient ribozymes-mediated operational code of tRNA aminoacylation not mutually contradicting but rather co-existing in harmony. Reviewers This article was reviewed by Eugene V. Koonin, Wentao Ma (nominated by Juergen Brosius) and Anthony Poole. PMID:21342520

A Bayesian network coding scheme for annotating biomedical information presented to genetic counseling clients.

PubMed

Green, Nancy

2005-04-01

We developed a Bayesian network coding scheme for annotating biomedical content in layperson-oriented clinical genetics documents. The coding scheme supports the representation of probabilistic and causal relationships among concepts in this domain, at a high enough level of abstraction to capture commonalities among genetic processes and their relationship to health. We are using the coding scheme to annotate a corpus of genetic counseling patient letters as part of the requirements analysis and knowledge acquisition phase of a natural language generation project. This paper describes the coding scheme and presents an evaluation of intercoder reliability for its tag set. In addition to giving examples of use of the coding scheme for analysis of discourse and linguistic features in this genre, we suggest other uses for it in analysis of layperson-oriented text and dialogue in medical communication.

Protocols development for security and privacy of radio frequency identification systems

NASA Astrophysics Data System (ADS)

Sabbagha, Fatin

There are benefits to adopting radio frequency identification (RFID) technology, although there are methods of attack that can compromise the system. This research determined how that may happen and what possible solutions can keep that from happening. Protocols were developed to implement better security. In addition, new topologies were developed to handle the problems of the key management. Previously proposed protocols focused on providing mutual authentication and privacy between readers and tags. However, those protocols are still vulnerable to be attacked. These protocols were analyzed and the disadvantages shown for each one. Previous works assumed that the channels between readers and the servers were secure. In the proposed protocols, a compromised reader is considered along with how to prevent tags from being read by that reader. The new protocols provide mutual authentication between readers and tags and, at the same time, remove the compromised reader from the system. Three protocols are proposed. In the first protocol, a mutual authentication is achieved and a compromised reader is not allowed in the network. In the second protocol, the number of times a reader contacts the server is reduced. The third protocol provides authentication and privacy between tags and readers using a trusted third party. The developed topology is implemented using python language and simulates work to check the efficiency regarding the processing time. The three protocols are implemented by writing codes in C language and then compiling them in MSP430. IAR Embedded workbench is used, which is an integrated development environment with the C/C++ compiler to generate a faster code and to debug the microcontroller. In summary, the goal of this research is to find solutions for the problems on previously proposed protocols, handle a compromised reader, and solve key management problems.

Whole-genome sequencing reveals a coding non-pathogenic variant tagging a non-coding pathogenic hexanucleotide repeat expansion in C9orf72 as cause of amyotrophic lateral sclerosis.

PubMed

Herdewyn, Sarah; Zhao, Hui; Moisse, Matthieu; Race, Valérie; Matthijs, Gert; Reumers, Joke; Kusters, Benno; Schelhaas, Helenius J; van den Berg, Leonard H; Goris, An; Robberecht, Wim; Lambrechts, Diether; Van Damme, Philip

2012-06-01

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) has a familial cause in 10% of patients. Despite significant advances in the genetics of the disease, many families remain unexplained. We performed whole-genome sequencing in five family members from a pedigree with autosomal-dominant classical ALS. A family-based elimination approach was used to identify novel coding variants segregating with the disease. This list of variants was effectively shortened by genotyping these variants in 2 additional unaffected family members and 1500 unrelated population-specific controls. A novel rare coding variant in SPAG8 on chromosome 9p13.3 segregated with the disease and was not observed in controls. Mutations in SPAG8 were not encountered in 34 other unexplained ALS pedigrees, including 1 with linkage to chromosome 9p13.2-23.3. The shared haplotype containing the SPAG8 variant in this small pedigree was 22.7 Mb and overlapped with the core 9p21 linkage locus for ALS and frontotemporal dementia. Based on differences in coverage depth of known variable tandem repeat regions between affected and non-affected family members, the shared haplotype was found to contain an expanded hexanucleotide (GGGGCC)(n) repeat in C9orf72 in the affected members. Our results demonstrate that rare coding variants identified by whole-genome sequencing can tag a shared haplotype containing a non-coding pathogenic mutation and that changes in coverage depth can be used to reveal tandem repeat expansions. It also confirms (GGGGCC)n repeat expansions in C9orf72 as a cause of familial ALS.

Measurement of fish movements at depths to 6000 m using a deep-ocean lander incorporating a short base-line sonar utilizing miniature code-activated transponder technology

NASA Astrophysics Data System (ADS)

Bagley, P. M.; Bradley, S.; Priede, I. G.; Gray, P.

1999-12-01

Most research on animal behaviour in the deep ocean (to depths of 6000 m) is restricted to the capture of dead specimens or viewing activity over small areas of the sea floor by means of cameras or submersibles. This paper describes the use of a miniature acoustic code-activated transponder (CAT) tag and short base-line sonar to track the movements of deep-sea fish in two dimensions over an area 1 km in diameter centred on a lander platform. The CAT tags and sonar are transported to the deep-sea floor by means of a subsea mooring which is ballasted so that it lands and remains on the sea floor for the duration of the tracking experiment (the lander). A description of the CAT, lander and short base-line sonar is given. Results are presented to illustrate the operation of the system.

Metal-ferroelectric-metal capacitor based persistent memory for electronic product code class-1 generation-2 uhf passive radio-frequency identification tag

NASA Astrophysics Data System (ADS)

Yoon, Bongno; Sung, Man Young; Yeon, Sujin; Oh, Hyun S.; Kwon, Yoonjoo; Kim, Chuljin; Kim, Kyung-Ho

2009-03-01

With the circuits using metal-ferroelectric-metal (MFM) capacitor, rf operational signal properties are almost the same or superior to those of polysilicon-insulator-polysilicon, metal-insulator-metal, and metal-oxide-semiconductor (MOS) capacitors. In electronic product code global class-1 generation-2 uhf radio-frequency identification (RFID) protocols, the MFM can play a crucial role in satisfying the specifications of the inventoried flag's persistence times (Tpt) for each session (S0-S3, SL). In this paper, we propose and design a new MFM capacitor based memory scheme of which persistence time for S1 flag is measured at 2.2 s as well as indefinite for S2, S3, and SL flags during the period of power-on. A ferroelectric random access memory embedded RFID tag chip is fabricated with an industry-standard complementary MOS process. The chip size is around 500×500 μm2 and the measured power consumption is about 10 μW.

Sugar versus fat: elimination of glycogen storage improves lipid accumulation in Yarrowia lipolytica.

PubMed

Bhutada, Govindprasad; Kavšcek, Martin; Ledesma-Amaro, Rodrigo; Thomas, Stéphane; Rechberger, Gerald N; Nicaud, Jean-Marc; Natter, Klaus

2017-05-01

Triacylglycerol (TAG) and glycogen are the two major metabolites for carbon storage in most eukaryotic organisms. We investigated the glycogen metabolism of the oleaginous Yarrowia lipolytica and found that this yeast accumulates up to 16% glycogen in its biomass. Assuming that elimination of glycogen synthesis would result in an improvement of lipid accumulation, we characterized and deleted the single gene coding for glycogen synthase, YlGSY1. The mutant was grown under lipogenic conditions with glucose and glycerol as substrates and we obtained up to 60% improvement in TAG accumulation compared to the wild-type strain. Additionally, YlGSY1 was deleted in a background that was already engineered for high lipid accumulation. In this obese background, TAG accumulation was also further increased. The highest lipid content of 52% was found after 3 days of cultivation in nitrogen-limited glycerol medium. Furthermore, we constructed mutants of Y. lipolytica and Saccharomyces cerevisiae that are deleted for both glycogen and TAG synthesis, demonstrating that the ability to store carbon is not essential. Overall, this work showed that glycogen synthesis is a competing pathway for TAG accumulation in oleaginous yeasts and that deletion of the glycogen synthase has beneficial effects on neutral lipid storage. © FEMS 2017.

Flexible ultra-wideband antenna incorporated with metamaterial structures: multiple notches for chipless RFID application

NASA Astrophysics Data System (ADS)

Jalil, M. E.; Rahim, M. K. A.; Samsuri, N. A.; Dewan, R.; Kamardin, K.

2017-01-01

A coplanar waveguide (CPW) ultra-wideband (UWB) antenna incorporated with metamaterial—split ring resonator structure—that operates from 3.0 to 12.0 GHz is proposed for chipless RFID tag. The 30 mm × 40 mm flexible chipless RFID tag is designed on the fleece substrate ( ɛ r = 1.35, thickness = 1 mm and tan δ = 0.025). A six-slotted modified complementary split ring resonator (MCSRR) is introduced into the ultra-wideband antenna to produce multiple band notches at 3.0, 4.0, 5.0, 6.0 and 7.0 GHz. The frequency shifting technique is introduced for designing a high-capacity chipless RFID tag with compact size. Each MCSRR is able to code in four different allocations (00, 01, 10 and 11). To achieve encoding of 10-bits data (10,234 number), six MCSRRs are proposed with three-slotted MCSRR in the radiator and three-slotted MCSRR in the ground plane.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

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

Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong

2016-01-08

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively boundmore » the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.« less

Expression and Production of SH2 Domain Proteins.

PubMed

Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

2017-01-01

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

PubMed

Hisada, Hiromoto; Tsutsumi, Hiroko; Ishida, Hiroki; Hata, Yoji

2013-01-01

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.

Identification of secreted bacterial proteins by noncanonical amino acid tagging

PubMed Central

Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.

2014-01-01

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637

Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point

PubMed Central

Jeon, Won Bae

2015-01-01

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value. PMID:20510014

Comparison of Zirconium Phosphonate-Modified Surfaces for Immobilizing Phosphopeptides and Phosphate-Tagged Proteins.

PubMed

Forato, Florian; Liu, Hao; Benoit, Roland; Fayon, Franck; Charlier, Cathy; Fateh, Amina; Defontaine, Alain; Tellier, Charles; Talham, Daniel R; Queffélec, Clémence; Bujoli, Bruno

2016-06-07

Different routes for preparing zirconium phosphonate-modified surfaces for immobilizing biomolecular probes are compared. Two chemical-modification approaches were explored to form self-assembled monolayers on commercially available primary amine-functionalized slides, and the resulting surfaces were compared to well-characterized zirconium phosphonate monolayer-modified supports prepared using Langmuir-Blodgett methods. When using POCl3 as the amine phosphorylating agent followed by treatment with zirconyl chloride, the result was not a zirconium-phosphonate monolayer, as commonly assumed in the literature, but rather the process gives adsorbed zirconium oxide/hydroxide species and to a lower extent adsorbed zirconium phosphate and/or phosphonate. Reactions giving rise to these products were modeled in homogeneous-phase studies. Nevertheless, each of the three modified surfaces effectively immobilized phosphopeptides and phosphopeptide tags fused to an affinity protein. Unexpectedly, the zirconium oxide/hydroxide modified surface, formed by treating the amine-coated slides with POCl3/Zr(4+), afforded better immobilization of the peptides and proteins and efficient capture of their targets.

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

PubMed Central

Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

2016-01-01

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae.

PubMed

Kim, Sujin; Bae, Sang-Jeong; Hahn, Ji-Sook

2016-04-07

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging.

Effects of Renibacterium salmoninarum on olfactory organs of Chinook salmon (Oncorhynchus tshawytscha) marked with coded wire tags

USGS Publications Warehouse

Elliott, Diane G.; Conway, Carla M.; Bruno, D.W.; Elliott, D.G.; Nowak, B.

2014-01-01

Bacterial kidney disease (BKD) caused by Renibacterium salmoninarum can cause significant morbidity and mortality in Chinook salmon (Oncorhynchus tshawytscha), particularly in Chinook salmon of the stream (spring) life history type, which migrate to sea as yearlings rather than subyearlings. R. salmoninarum can be transmitted vertically from the female parent to the progeny in association with the egg, as well as horizontally from fish to fish. This study was conducted as part of a research project to investigate whether the prevalence and intensity of R. salmoninarum infections in adult spring Chinook salmon could affect the survival and pathogen prevalence and intensity in their progeny (Pascho et al., 1991, 1993; Elliott et al., 1995). Fish from two brood years (1988 and 1989) were reared at Dworshak National Fish Hatchery (Idaho, USA) for about 1-1/2 years, released as yearling smolts, and allowed to migrate to the Pacific Ocean for maturation. The majority of progeny fish were marked with coded wire tags (CWTs) about 4 months before they were released from the hatchery so that adult returns could be monitored. The CWTs were implanted in the snouts of the fish by an experienced team of fish markers using automated wire-tagging machines. The intended placement site was the cartilage, skeletal muscle or loose connective tissue of the snout.

Bar codes and intrinsic-surface-roughness tag: Accurate and low-cost accountability for CFE. [Conventional force equipment (CFE)

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

DeVolpi, A.; Palm, R.

CFE poses a number of verification challenges that could be met in part by an accurate and low-cost means of aiding in accountability of treaty-limited equipment. Although the treaty as signed does not explicitly call for the use of tags, there is a provision for recording serial numbers'' and placing special marks'' on equipment subject to reduction. There are approximately 150,000 residual items to be tracked for CFE-I, about half for each alliance of state parties. These highly mobile items are subject to complex treaty limitations: deployment limits and zones, ceilings subceilings, holdings and allowances. There are controls and requirementsmore » for storage, conversion, and reduction. In addition, there are national security concerns regarding modernization and mobilization capability. As written into the treaty, a heavy reliance has been placed on human inspectors for CFE verification. Inspectors will mostly make visual observations and photographs as the means of monitoring compliance; these observations can be recorded by handwriting or keyed into a laptop computer. CFE is now less a treaty between two alliances than a treaty among 22 state parties, with inspection data an reports to be shared with each party in the official languages designated by CSCE. One of the potential roles for bar-coded tags would be to provide a universal, exchangable, computer-compatible language for tracking TLE. 10 figs.« less

Microbeads display of proteins using emulsion PCR and cell-free protein synthesis.

PubMed

Gan, Rui; Yamanaka, Yumiko; Kojima, Takaaki; Nakano, Hideo

2008-01-01

We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. A PCR mixture containing streptavidin-coated microbeads was compartmentalized by water-in-oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead-linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti-glutathione S-transferase (GST) antibody. The pool of beads was then subjected to cell-free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)-fused and FLAG tag-fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)-labeled anti-His6 (C-term) antibody, the beads with the His6 gene were enriched 917-fold in a single-round screening by using flow cytometry. A library with a theoretical diversity of 10(6) was constructed by randomizing the middle four residues of the His6 tag. After a two-round screening, the randomized sequences were substantially converged to peptide-encoding sequences recognized by the anti-His6 antibody.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

PubMed

Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

2009-11-01

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

PubMed

Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2015-04-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. © 2015 The Authors.

Quantitative proteomic analysis of CMS-related changes in Honglian CMS rice anther.

PubMed

Sun, Qingping; Hu, Chaofeng; Hu, Jun; Li, Shaoqing; Zhu, Yingguo

2009-10-01

Honglian (HL) cytoplasmic male sterility (CMS) is one of the rice CMS types and has been widely used in hybrid rice production in China. The CMS line (Yuetai A, YTA) has a Yuetai B (maintainer line, YTB) nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of Yuetai B. The fertility of hybrid (HL-6) was restored by restorer gene in nuclear genome of restorer line (9311). We used isotope-code affinity tag (ICAT) technology to perform the protein profiling of uninucleate stage rice anther and identify the CMS-HL related proteins. Two separate ICAT analyses were performed in this study: (1) anthers from YTA versus anthers from YTB, and (2) anthers from YTA versus anthers from HL-6. Based on the two analyses, a total of 97 unique proteins were identified and quantified in uninucleate stage rice anther under the error rate of less than 10%, of which eight proteins showed abundance changes of at least twofold between YTA and YTB. Triosephosphate isomerase, fructokinase II, DNA-binding protein GBP16 and ribosomal protein L3B were over-expressed in YTB, while oligopeptide transporter, floral organ regulator 1, kinase and S-adenosyl-L: -methionine synthetase were over-expressed in YTA. Reduction of the proteins associated with energy production and lesser ATP equivalents detected in CMS anther indicated that the low level of energy production played an important role in inducing CMS-HL.

Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.

PubMed Central

Kanugula, Sreenivas; Pegg, Anthony E

2003-01-01

AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy. PMID:12892560

Thiol-based Redox Proteins in Brassica napus Guard Cell Abscisic Acid and Methyl Jasmonate Signaling

PubMed Central

Zhu, Mengmeng; Zhu, Ning; Song, Wen-yuan; Harmon, Alice C.; Assmann, Sarah M.; Chen, Sixue

2014-01-01

SUMMARY Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in different physiological processes. Little is known, however, about redox sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis (DIGE) and isotope-coded affinity tag (ICAT). In total, 65 and 118 potential redox responsive proteins were identified in ABA and MeJA treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of energy, stress and defense, and metabolism. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA and MeJA treated samples. A total of 44 cysteines was mapped in all the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a SNRK2 kinase and an isopropylmalate dehydrogenase were confirmed to be redox regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in guard cell ABA and MeJA signal transduction. PMID:24580573

A New Operating System for Security Tagged Architecture Hardware in Support of Multiple Independent Levels of Security (MILS) Compliant System

DTIC Science & Technology

2014-04-01

important data structures of RTEMS are introduced. Section 3.2.2 discusses the problems we found in RTEMS that may cause security vulnerabilities...the important data structures in RTEMS: Object, which is a critical data structure in the SCORE, tasks threads. Approved for Public Release...these important system codes. The example code shows a possibility that a user can delete a system thread. Therefore, in order to protect system

Immuno-affinity Capture Followed by TMPP N-Terminus Tagging to Study Catabolism of Therapeutic Proteins.

PubMed

Kullolli, Majlinda; Rock, Dan A; Ma, Ji

2017-02-03

Characterization of in vitro and in vivo catabolism of therapeutic proteins has increasingly become an integral part of discovery and development process for novel proteins. Unambiguous and efficient identification of catabolites can not only facilitate accurate understanding of pharmacokinetic profiles of drug candidates, but also enables follow up protein engineering to generate more catabolically stable molecules with improved properties (pharmacokinetics and pharmacodynamics). Immunoaffinity capture (IC) followed by top-down intact protein analysis using either matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry analysis have been the primary methods of choice for catabolite identification. However, the sensitivity and efficiency of these methods is not always sufficient for characterization of novel proteins from complex biomatrices such as plasma or serum. In this study a novel bottom-up targeted protein workflow was optimized for analysis of proteolytic degradation of therapeutic proteins. Selective and sensitive tagging of the alpha-amine at the N-terminus of proteins of interest was performed by immunoaffinity capture of therapeutic protein and its catabolites followed by on-bead succinimidyloxycarbonylmethyl tri-(2,4,6-trimethoxyphenyl N-terminus (TMPP-NTT) tagging. The positively charged hydrophobic TMPP tag facilitates unambiguous sequence identification of all N-terminus peptides from complex tryptic digestion samples via data dependent liquid chromatgraphy-tandem mass spectroscopy. Utility of the workflow was illustrated by definitive analysis of in vitro catabolic profile of neurotensin human Fc (NTs-huFc) protein in mouse serum. The results from this study demonstrated that the IC-TMPP-NTT workflow is a simple and efficient method for catabolite formation in therapeutic proteins.

Novel high-performance purification protocol of recombinant CNBP suitable for biochemical and biophysical characterization.

PubMed

Challier, Emilse; Lisa, María-Natalia; Nerli, Bibiana B; Calcaterra, Nora B; Armas, Pablo

2014-01-01

Cellular nucleic acid binding protein (CNBP) is a highly conserved multi-zinc knuckle protein that enhances c-MYC expression, is related to certain human muscular diseases and is required for proper rostral head development. CNBP binds to single-stranded DNA (ssDNA) and RNA and acts as nucleic acid chaperone. Despite the advances made concerning CNBP biological roles, a full knowledge about the structure-function relationship has not yet been achieved, likely due to difficulty in obtaining pure and tag-free CNBP. Here, we report a fast, simple, reproducible, and high-performance expression and purification protocol that provides recombinant tag-free CNBP from Escherichia coli cultures. We determined that tag-free CNBP binds its molecular targets with higher affinity than tagged-CNBP. Furthermore, fluorescence spectroscopy revealed the presence of a unique and conserved tryptophan, which is exposed to the solvent and involved, directly or indirectly, in nucleic acid binding. Size-exclusion HPLC revealed that CNBP forms homodimers independently of nucleic acid binding and coexist with monomers as non-interconvertible forms or in slow equilibrium. Circular dichroism spectroscopy showed that CNBP has a secondary structure dominated by random-coil and β-sheet coincident with the sequence-predicted repetitive zinc knuckles motifs, which folding is required for CNBP structural stability and biochemical activity. CNBP structural stability increased in the presence of single-stranded nucleic acid targets similar to other unstructured nucleic acid chaperones. Altogether, data suggest that CNBP is a flexible protein with interspersed structured zinc knuckles, and acquires a more rigid structure upon nucleic acid binding. Copyright © 2013 Elsevier Inc. All rights reserved.

Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes

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

Martin-Diaconescu, Vlad; Joseph, Crisjoe A.; Boer, Jodi L.

Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donormore » ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.« less

Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP

NASA Technical Reports Server (NTRS)

Mullins, D. W., Jr.; Lacey, J. C., Jr.

1981-01-01

The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

Research Advances

ERIC Educational Resources Information Center

King, Angela G.

2004-01-01

Nanotechnology are employed by researchers at Northwestern University to develop a method of labeling disease markers present in blood with unique DNA tags they have dubbed "bio-bar-codes". The preparation of nanoparticle and magnetic microparticle probes and a nanoparticle-based PSR-less DNA amplification scheme are involved by the DNA-BCA assay.

MCNP calculations for container inspection with tagged neutrons

NASA Astrophysics Data System (ADS)

Boghen, G.; Donzella, A.; Filippini, V.; Fontana, A.; Lunardon, M.; Moretto, S.; Pesente, S.; Zenoni, A.

2005-12-01

We are developing an innovative tagged neutrons inspection system (TNIS) for cargo containers: the system will allow us to assay the chemical composition of suspect objects, previously identified by a standard X-ray radiography. The operation of the system is extensively being simulated by using the MCNP Monte Carlo code to study different inspection geometries, cargo loads and hidden threat materials. Preliminary simulations evaluating the signal and the signal over background ratio expected as a function of the system parameters are presented. The results for a selection of cases are briefly discussed and demonstrate that the system can operate successfully in different filling conditions.

Design and expression of recombinant toxins from Mexican scorpions of the genus Centruroides for production of antivenoms.

PubMed

Jiménez-Vargas, J M; Quintero-Hernández, V; González-Morales, L; Ortiz, E; Possani, L D

2017-03-15

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD 50 ) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD 50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 μL of serum from any of the immunized rabbits were enough to neutralize three LD 50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Digitally Enhanced Heterodyne Interferometry

NASA Technical Reports Server (NTRS)

Shaddock, Daniel; Ware, Brent; Lay, Oliver; Dubovitsky, Serge

2010-01-01

Spurious interference limits the performance of many interferometric measurements. Digitally enhanced interferometry (DEI) improves measurement sensitivity by augmenting conventional heterodyne interferometry with pseudo-random noise (PRN) code phase modulation. DEI effectively changes the measurement problem from one of hardware (optics, electronics), which may deteriorate over time, to one of software (modulation, digital signal processing), which does not. DEI isolates interferometric signals based on their delay. Interferometric signals are effectively time-tagged by phase-modulating the laser source with a PRN code. DEI improves measurement sensitivity by exploiting the autocorrelation properties of the PRN to isolate only the signal of interest and reject spurious interference. The properties of the PRN code determine the degree of isolation.

Annual Coded Wire Tag Program; Oregon Stock Assessment, 2000 Annual Report.

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

Lewis, Mark; Mallette, Christine; Murray, William

2002-03-01

This annual report is in fulfillment of contract obligations with Bonneville Power Administration which is the funding source for the Oregon Department of Fish and Wildlife's Annual Stock Assessment - Coded Wire Tag Program (ODFW) Project. Tule stock fall chinook were caught primarily in British Columbia and Washington ocean, and Columbia Basin fisheries. Up-river bright stock fall chinook contributed primarily to Alaska and British Columbia ocean commercial, Columbia Basin gillnet and freshwater sport fisheries. Contribution of Rogue stock fall chinook released in the lower Columbia River occurred primarily in Oregon ocean commercial, Columbia Basin gillnet and freshwater sport fisheries. Willamettemore » stock spring chinook contributed primarily to Alaska and British Columbia ocean, and Columbia Basin sport fisheries. Willamette stock spring chinook released by CEDC contributed to similar ocean fisheries, but had much higher catch in Columbia Basin gillnet fisheries than the same stocks released in the Willamette Basin. Up-river stocks of spring chinook contributed almost exclusively to Columbia Basin fisheries. The up-river stocks of Columbia River summer steelhead contributed almost exclusively to the Columbia Basin gillnet and freshwater sport fisheries. Coho ocean fisheries from Washington to California were closed or very limited from 1994 through 1999 (1991 through 1996 broods). This has resulted in a lower percent of catch in Washington, Oregon and California ocean fisheries, and a higher percent of catch in Alaska and British Columbia ocean and Columbia Basin freshwater fisheries. Coho stocks released by ODFW below Bonneville Dam were caught mainly in Oregon, Washington, and British Columbia ocean, Columbia Gillnet and freshwater sport fisheries. Coho stocks released in the Klaskanine River and Youngs Bay area had similar ocean catch distributions, but a much higher percent catch in gillnet fisheries than the other coho releases. Ocean catch distribution of coho stocks released above Bonneville Dam was similar to the other coho groups. However, they had a higher percent catch in gillnet fisheries above Bonneville Dam than coho released below the dam. Survival rates of salmon and steelhead are influenced, not only by factors in the hatchery (disease, density, diet, size and time of release) but also by environmental factors in the river and ocean. These environmental factors are influenced by large scale oceanic and weather patterns such as El Nino. Changes in rearing conditions in the hatchery do impact survival, however, these can be offset by impacts caused by environmental factors. Coho salmon released in the Columbia River generally experience better survival rates when released later in the spring. However, for the 1990 brood year June releases of Columbia River coho had much lower survival than May releases, for all ODFW hatcheries. In general survival of ODFW Columbia River hatchery coho has declined to low levels in recent years. Preliminary results from the evaluation of Visual Implant Elastomer (VIE) tags showed tagging rate and pre-release tag retention improved from the first to second years of tagging. Tagging rate remained identical from 1999 to 2000 while pre-release tag retention dropped to 95%. Returning jack and adult salmon were sampled for CWT and VIE tags in the fall of 2000. Of 606 adults recovered at Sandy Fish Hatchery in 2000, only 1 or 0.2%, retained their VIE tag. Of 36 jacks recovered in 2000, 13 or 36.1% retained their VIE tag.« less

Image authentication using distributed source coding.

PubMed

Lin, Yao-Chung; Varodayan, David; Girod, Bernd

2012-01-01

We present a novel approach using distributed source coding for image authentication. The key idea is to provide a Slepian-Wolf encoded quantized image projection as authentication data. This version can be correctly decoded with the help of an authentic image as side information. Distributed source coding provides the desired robustness against legitimate variations while detecting illegitimate modification. The decoder incorporating expectation maximization algorithms can authenticate images which have undergone contrast, brightness, and affine warping adjustments. Our authentication system also offers tampering localization by using the sum-product algorithm.

Parallel tiled Nussinov RNA folding loop nest generated using both dependence graph transitive closure and loop skewing.

PubMed

Palkowski, Marek; Bielecki, Wlodzimierz

2017-06-02

RNA secondary structure prediction is a compute intensive task that lies at the core of several search algorithms in bioinformatics. Fortunately, the RNA folding approaches, such as the Nussinov base pair maximization, involve mathematical operations over affine control loops whose iteration space can be represented by the polyhedral model. Polyhedral compilation techniques have proven to be a powerful tool for optimization of dense array codes. However, classical affine loop nest transformations used with these techniques do not optimize effectively codes of dynamic programming of RNA structure predictions. The purpose of this paper is to present a novel approach allowing for generation of a parallel tiled Nussinov RNA loop nest exposing significantly higher performance than that of known related code. This effect is achieved due to improving code locality and calculation parallelization. In order to improve code locality, we apply our previously published technique of automatic loop nest tiling to all the three loops of the Nussinov loop nest. This approach first forms original rectangular 3D tiles and then corrects them to establish their validity by means of applying the transitive closure of a dependence graph. To produce parallel code, we apply the loop skewing technique to a tiled Nussinov loop nest. The technique is implemented as a part of the publicly available polyhedral source-to-source TRACO compiler. Generated code was run on modern Intel multi-core processors and coprocessors. We present the speed-up factor of generated Nussinov RNA parallel code and demonstrate that it is considerably faster than related codes in which only the two outer loops of the Nussinov loop nest are tiled.

Site Specific Discrete PEGylation of 124I-Labeled mCC49 Fab′ Fragments Improves Tumor MicroPET/CT Imaging in Mice

PubMed Central

Ding, Haiming; Carlton, Michelle M.; Povoski, Stephen P.; Milum, Keisha; Kumar, Krishan; Kothandaraman, Shankaran; Hinkle, George H.; Colcher, David; Brody, Rich; Davis, Paul D.; Pokora, Alex; Phelps, Mitchell; Martin, Edward W.; Tweedle, Michael F.

2014-01-01

The tumor-associated glycoprotein-72 (TAG-72) antigen is highly overexpressed in various human adenocarcinomas and anti-TAG-72 monoclonal antibodies, and fragments are therefore useful as pharmaceutical targeting vectors. In this study, we investigated the effects of site-specific PEGylation with MW 2–4 kDa discrete, branched PEGylation reagents on mCC49 Fab′ (MW 50 kDa) via in vitro TAG72 binding, and in vivo blood clearance kinetics, biodistribution, and mouse tumor microPET/CT imaging. mCC49Fab′ (Fab′-NEM) was conjugated at a hinge region cysteine with maleimide-dPEG12-(dPEG24COOH)3 acid (Mal-dPEG-A), maleimide-dPEG12-(dPEG12COOH)3 acid (Mal-dPEG-B), or maleimide-dPEG12-(m-dPEG24)3 (Mal-dPEG-C), and then radiolabeled with iodine-124 (124I) in vitro radioligand binding assays and in vivo studies used TAG-72 expressing LS174T human colon carcinoma cells and xenograft mouse tumors. Conjugation of mCC49Fab′ with Mal-dPEG-A (Fab′-A) reduced the binding affinity of the non PEGylated Fab′ by 30%; however, in vivo, Fab′-A significantly lengthened the blood retention vs Fab′-NEM (47.5 vs 28.1%/ID at 1 h, 25.1 vs 8.4%/ID at 5 h, p < 0.01), showed excellent tumor to background, better microPET/CT images due to higher tumor accumulation, and increased tumor concentration in excised tissues at 72 h by 130% (5.09 ± 0.83 vs 3.83 ± 1.50%ID/g, p < 0.05). Despite the strong similarity of the three PEGylation reagents, PEGylation with Mal-dPEG-B or -C reduced the in vitro binding affinity of Fab′-NEM by 70%, blood retention, microPET/CT imaging tumor signal intensity, and residual 72 h tumor concentration by 49% (3.83 ± 1.50 vs 1.97 ± 0.29%ID/g, p < 0.05) and 63% (3.83 ± 1.50 vs 1.42 ± 0.35%ID/g, p < 0.05), respectively. We conclude that remarkably subtle changes in the structure of the PEGylation reagent can create significantly altered biologic behavior. Further study is warranted of conjugates of the triple branched, negatively charged Mal-dPEG-A. PMID:24175669

Toward Fast and Accurate Binding Affinity Prediction with pmemdGTI: An Efficient Implementation of GPU-Accelerated Thermodynamic Integration.

PubMed

Lee, Tai-Sung; Hu, Yuan; Sherborne, Brad; Guo, Zhuyan; York, Darrin M

2017-07-11

We report the implementation of the thermodynamic integration method on the pmemd module of the AMBER 16 package on GPUs (pmemdGTI). The pmemdGTI code typically delivers over 2 orders of magnitude of speed-up relative to a single CPU core for the calculation of ligand-protein binding affinities with no statistically significant numerical differences and thus provides a powerful new tool for drug discovery applications.

HowTo - Easy use of global unique identifier

NASA Astrophysics Data System (ADS)

Czerniak, A.; Fleischer, D.; Schirnick, C.

2013-12-01

The GEOMAR sample- and core repository covers several thousands of samples and cores and was collected over the last decades. In the actual project, we bring this collection up to the new generation and tag every sample and core with a unique identifier, in our case the International Geo Sample Number (ISGN). This work is done with our digital Ink and hand writing recognition implementation. The Smart Pen technology was save time and resources to record the information on every sample or core. In the procedure of recording, there are several steps systematical are done: 1. Getting all information about the core or sample, such as cruise number, responsible person and so on. 2. Tag with unique identifiers, in our case a QR-Code. 3. Wrote down the location of sample or core. After transmitting the information from Smart Pen, actually via USB but wireless is a choice too, into our server infrastructure the link to other information began. As it linked in our Virtual Research Environment (VRE) with the unique identifier (ISGN) sample or core can be located and the QR-Code was simply linked back from core or sample to ISGN with additional scientific information. On the QR-Code all important information are on it and it was simple to produce thousand of it.

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

PubMed

Mainali, Dipak; Smith, Emily A

2013-04-01

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

Salmon Supplementation Studies in Idaho Rivers; Field Activities Conducted on Clear and Pete King Creeks, 2002 Annual Report.

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

Bretz, Justin K.; Olson, Jill M.

2003-03-01

In 2002 the Idaho Fisheries Resource Office continued working as a cooperator on the Salmon Supplementation Studies in Idaho Rivers (ISS) project on Pete King and Clear creeks. Data relating to supplementation treatment releases, juvenile sampling, juvenile PIT tagging, broodstock spawning and rearing, spawning ground surveys, and snorkel surveys were used to evaluate the project data points and augment past data. Supplementation treatments included the release of 51,329 left ventral-clipped smolts into Clear Creek (750 were PIT tagged), and 12,000 unmarked coded-wire tagged parr into Pete King Creek (998 were PIT tagged). Using juvenile collection methods, Idaho Fisheries Resource Officemore » staff PIT tagged and released 579 naturally produced spring chinook juveniles in Clear Creek, and 54 on Pete King Creek, for minimum survival estimates to Lower Granite Dam. For Clear Creek, minimum survival estimates to Lower Granite Dam of hatchery produced supplementation and naturally produced PIT tagged smolts, were 36.0%, and 53.1%, respectively. For Pete King Creek, minimum survival estimates to Lower Granite Dam, of hatchery produced supplementation smolts and naturally produced smolts PIT tagged as parr and presmolts, were 18.8%, and 8.3%, respectively. Adults collected for broodstock in 2002 represented the final adult broodstock group collected for the ISS project. Twenty-six ventral clipped, and 28 natural adult spring chinook were transported above the weir. Monitoring and evaluation of spawning success was continued on Clear and Pete King creeks. A total of 69 redds were counted and 79 carcasses were recovered on Clear Creek. Two redds were observed and no carcasses were collected on Pete King Creek.« less

Affinity-aware checkpoint restart

DOE PAGES

Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

2014-12-08

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Affinity-aware checkpoint restart

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

Saini, Ajay; Rezaei, Arash; Mueller, Frank

Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

PubMed Central

2011-01-01

Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge. PMID:21824404

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

PubMed

Chen, WeiYue; Avezov, Edward; Schlachter, Simon C; Gielen, Fabrice; Laine, Romain F; Harding, Heather P; Hollfelder, Florian; Ron, David; Kaminski, Clemens F

2015-03-10

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Application of histone modification-specific interaction domains as an alternative to antibodies.

PubMed

Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul; Jeltsch, Albert

2014-11-01

Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. © 2014 Kungulovski et al.; Published by Cold Spring Harbor Laboratory Press.

Probing Cocaine-Antibody Interactions in Buffer and Human Serum

PubMed Central

Ramakrishnan, Muthu; Alves De Melo, Fernando; Kinsey, Berma M.; Ladbury, John E.; Kosten, Thomas R.; Orson, Frank M.

2012-01-01

Background Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum. Results MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20–50% human serum compared with that in saline buffer. A similar trend was also found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies. Conclusions High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide valuable information for characterization of their interactions and thermodynamic properties. In addition MST measurements of antibody affinity in the presence of biological fluids will provide a better opportunity to make reliable decisions and facilitate the design of cocaine vaccines and immunization conditions. The methods should be more widely adopted in characterization of antibody complexes. PMID:22859949

Effects of routine handling and tagging procedures on physiological stress responses in juvenile chinook salmon

USGS Publications Warehouse

Sharpe, C.S.; Thompson, D.A.; Blankenship, H.L.; Schreck, C.B.

1998-01-01

Juvenile chinook salmon Oncorhynchus tshawytscha were subjected to handling and tagging protocols typical of normal hatchery operations and monitored for their physiological response to stress. Treatments included coded-wire-tagging, counting, ventral fin clipping, adipose fin clipping, and a procedure simulating a pond split. Treatment fish were also subjected to a standardized stress challenge (1 h confinement) to evaluate their ability to deal with disturbances subsequent to a handling or tagging procedure. Circulating levels of cortisol and glucose were used as indicators of stress. Each of the treatments elicited very similar responses among treatment groups. Cortisol increased from resting levels of about 20 ng/mL to about 90 ng/mL by 1 h poststress and returned to near resting levels by 8 h poststress. Glucose levels increased from 50 mg/dL to about 80 mg/dL by 1 h poststress and remained elevated for much of the experiment. The cortisol and glucose responses to the confinement stress did not differ over time or among treatments. However, the confinement stress results do suggest a small but significant cumulative response, indicating small residual effects of the original handling protocols. No deaths were noted among treatment groups.

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

PubMed Central

Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G.

2017-01-01

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays. PMID:28607052

Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

PubMed

Thomson, Joshua J; Withey, Jeffrey H

2014-11-01

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.

PubMed

Konziase, Benetode

2015-08-01

We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa. Copyright © 2015 Elsevier Inc. All rights reserved.

Near-Field Chipless Radio-Frequency Identification (RFID) Sensing and Identification System with Switching Reading.

PubMed

Paredes, Ferran; Herrojo, Cristian; Mata-Contreras, Javier; Moras, Miquel; Núñez, Alba; Ramon, Eloi; Martín, Ferran

2018-04-09

A chipless radio-frequency identification (chipless-RFID) and sensing system, where tags are read by proximity (near-field) through a switch, is presented. The tags consist of a set of identical resonant elements (split-ring resonators or SRRs), printed or etched at predefined and equidistant positions, forming a linear chain, each SRR providing a bit of information. The logic state ('1' or '0') associated with each resonator depends on whether it is present or not in the predefined position. The reader is an array of power splitters used to feed a set of SRR-loaded transmission lines (in equal number to the number of resonant elements, or bits, of the tag). The feeding (interrogation) signal is a harmonic (single-tone) signal tuned to a frequency in the vicinity of the fundamental resonance of the SRRs. The set of SRR-loaded lines must be designed so that the corresponding SRRs are in perfect alignment with the SRRs of the tag, provided the tag is positioned on top of the reader. Thus, in a reading operation, as long as the tag is very close to the reader, the SRRs of the tag modify (decrease) the transmission coefficient of the corresponding reader line (through electromagnetic coupling between both SRRs), and the amplitude of the output signal is severely reduced. Therefore, the identification (ID) code of the tag is contained in the amplitudes of the output signals of the SRR-loaded lines, which can be inferred sequentially by means of a switching system. Unlike previous chipless-RFID systems based on near-field and sequential bit reading, the tags in the proposed system can be merely positioned on top of the reader, conveniently aligned, without the need to mechanically place them across the reader. Since tag reading is only possible if the tag is very close to the reader, this system can be also used as a proximity sensor with applications such as target identification. The proposed chipless-RFID and sensing approach is validated by reading a designed 4-bit tag. For identification purposes, this system is of special interest in applications where a low number of bits suffice, and tag reading by proximity is acceptable (or even convenient). Applications mostly related to secure paper, particularly involving a limited number of items (e.g., exams, ballots, etc.), in order to provide authenticity and avoid counterfeiting, are envisaged. As a proximity sensor, the system may be of use in detecting and distinguishing different targets in applications such as smart packaging.

Near-Field Chipless Radio-Frequency Identification (RFID) Sensing and Identification System with Switching Reading

PubMed Central

Mata-Contreras, Javier; Moras, Miquel; Ramon, Eloi; Martín, Ferran

2018-01-01

A chipless radio-frequency identification (chipless-RFID) and sensing system, where tags are read by proximity (near-field) through a switch, is presented. The tags consist of a set of identical resonant elements (split-ring resonators or SRRs), printed or etched at predefined and equidistant positions, forming a linear chain, each SRR providing a bit of information. The logic state (‘1’ or ‘0’) associated with each resonator depends on whether it is present or not in the predefined position. The reader is an array of power splitters used to feed a set of SRR-loaded transmission lines (in equal number to the number of resonant elements, or bits, of the tag). The feeding (interrogation) signal is a harmonic (single-tone) signal tuned to a frequency in the vicinity of the fundamental resonance of the SRRs. The set of SRR-loaded lines must be designed so that the corresponding SRRs are in perfect alignment with the SRRs of the tag, provided the tag is positioned on top of the reader. Thus, in a reading operation, as long as the tag is very close to the reader, the SRRs of the tag modify (decrease) the transmission coefficient of the corresponding reader line (through electromagnetic coupling between both SRRs), and the amplitude of the output signal is severely reduced. Therefore, the identification (ID) code of the tag is contained in the amplitudes of the output signals of the SRR-loaded lines, which can be inferred sequentially by means of a switching system. Unlike previous chipless-RFID systems based on near-field and sequential bit reading, the tags in the proposed system can be merely positioned on top of the reader, conveniently aligned, without the need to mechanically place them across the reader. Since tag reading is only possible if the tag is very close to the reader, this system can be also used as a proximity sensor with applications such as target identification. The proposed chipless-RFID and sensing approach is validated by reading a designed 4-bit tag. For identification purposes, this system is of special interest in applications where a low number of bits suffice, and tag reading by proximity is acceptable (or even convenient). Applications mostly related to secure paper, particularly involving a limited number of items (e.g., exams, ballots, etc.), in order to provide authenticity and avoid counterfeiting, are envisaged. As a proximity sensor, the system may be of use in detecting and distinguishing different targets in applications such as smart packaging. PMID:29642560

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

PubMed

Manuel-Cabrera, Carlos Alberto; Vallejo-Cardona, Alba Adriana; Padilla-Camberos, Eduardo; Hernández-Gutiérrez, Rodolfo; Herrera-Rodríguez, Sara Elisa; Gutiérrez-Ortega, Abel

2016-11-29

Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

Preparation of silica coated cobalt ferrite magnetic nanoparticles for the purification of histidine-tagged proteins

NASA Astrophysics Data System (ADS)

Aygar, Gülfem; Kaya, Murat; Özkan, Necati; Kocabıyık, Semra; Volkan, Mürvet

2015-12-01

Surface modified cobalt ferrite (CoFe2O4) nanoparticles containing Ni-NTA affinity group were synthesized and used for the separation of histidine tag proteins from the complex matrices through the use of imidazole side chains of histidine molecules. Firstly, CoFe2O4 nanoparticles with a narrow size distribution were prepared in an aqueous solution using the controlled co-precipitation method. In order to obtain small CoFe2O4 agglomerates, oleic acid and sodium chloride were used as dispersants. The CoFe2O4 particles were coated with silica and subsequently the surface of these silica coated particles (SiO2-CoFe2O4) was modified by amine (NH2) groups in order to add further functional groups on the silica shell. Then, carboxyl (-COOH) functional groups were added to the SiO2-CoFe2O4 magnetic nanoparticles through the NH2 groups. After that Nα,Nα-Bis(carboxymethyl)-L-lysine hydrate (NTA) was attached to carboxyl ends of the structure. Finally, the surface modified nanoparticles were labeled with nickel (Ni) (II) ions. Furthermore, the modified SiO2-CoFe2O4 magnetic nanoparticles were utilized as a new system that allows purification of the N-terminal His-tagged recombinant small heat shock protein, Tpv-sHSP 14.3.

Unique self-assembly properties of a bridge-shaped protein dimer with quantum dots

NASA Astrophysics Data System (ADS)

Wang, Jianhao; Jiang, Pengju; Gao, Liqian; Yu, Yongsheng; Lu, Yao; Qiu, Lin; Wang, Cheli; Xia, Jiang

2013-09-01

How protein-protein interaction affects protein-nanoparticle self-assembly is the key to the understanding of biomolecular coating of nanoparticle in biological fluids. However, the relationship between protein shape and its interaction with nanoparticles is still under-exploited because of lack of a well-conceived binding system and a method to detect the subtle change in the protein-nanoparticle assemblies. Noticing this unresolved need, we cloned and expressed a His-tagged SpeA protein that adopts a bridge-shaped dimer structure, and utilized a high-resolution capillary electrophoresis method to monitor assembly formation between the protein and quantum dots (QDs, 5 nm in diameter). We observed that the bridge-shaped structure rendered a low SpeA:QD stoichiometry at saturation. Also, close monitoring of imidazole (Im) displacement of surface-bound protein revealed a unique two-step process. High-concentration Im could displace surface-bound SpeA protein and form a transient QD-protein intermediate, through a kinetically controlled displacement process. An affinity-driven equilibrium step then followed, resulting in re-assembling of the QD-protein complex in about 1 h. Through a temporarily formed intermediate, Im causes a rearrangement of His-tagged proteins on the surface. Thus, our work showcases that the synergistic interplay between QD-His-tag interaction and protein-protein interaction can result in unique properties of protein-nanoparticle assembly for the first time.

Automatic Data Traffic Control on DSM Architecture

NASA Technical Reports Server (NTRS)

Frumkin, Michael; Jin, Hao-Qiang; Yan, Jerry; Kwak, Dochan (Technical Monitor)

2000-01-01

We study data traffic on distributed shared memory machines and conclude that data placement and grouping improve performance of scientific codes. We present several methods which user can employ to improve data traffic in his code. We report on implementation of a tool which detects the code fragments causing data congestions and advises user on improvements of data routing in these fragments. The capabilities of the tool include deduction of data alignment and affinity from the source code; detection of the code constructs having abnormally high cache or TLB misses; generation of data placement constructs. We demonstrate the capabilities of the tool on experiments with NAS parallel benchmarks and with a simple computational fluid dynamics application ARC3D.

Tuning iteration space slicing based tiled multi-core code implementing Nussinov's RNA folding.

PubMed

Palkowski, Marek; Bielecki, Wlodzimierz

2018-01-15

RNA folding is an ongoing compute-intensive task of bioinformatics. Parallelization and improving code locality for this kind of algorithms is one of the most relevant areas in computational biology. Fortunately, RNA secondary structure approaches, such as Nussinov's recurrence, involve mathematical operations over affine control loops whose iteration space can be represented by the polyhedral model. This allows us to apply powerful polyhedral compilation techniques based on the transitive closure of dependence graphs to generate parallel tiled code implementing Nussinov's RNA folding. Such techniques are within the iteration space slicing framework - the transitive dependences are applied to the statement instances of interest to produce valid tiles. The main problem at generating parallel tiled code is defining a proper tile size and tile dimension which impact parallelism degree and code locality. To choose the best tile size and tile dimension, we first construct parallel parametric tiled code (parameters are variables defining tile size). With this purpose, we first generate two nonparametric tiled codes with different fixed tile sizes but with the same code structure and then derive a general affine model, which describes all integer factors available in expressions of those codes. Using this model and known integer factors present in the mentioned expressions (they define the left-hand side of the model), we find unknown integers in this model for each integer factor available in the same fixed tiled code position and replace in this code expressions, including integer factors, with those including parameters. Then we use this parallel parametric tiled code to implement the well-known tile size selection (TSS) technique, which allows us to discover in a given search space the best tile size and tile dimension maximizing target code performance. For a given search space, the presented approach allows us to choose the best tile size and tile dimension in parallel tiled code implementing Nussinov's RNA folding. Experimental results, received on modern Intel multi-core processors, demonstrate that this code outperforms known closely related implementations when the length of RNA strands is bigger than 2500.

CSTminer: a web tool for the identification of coding and noncoding conserved sequence tags through cross-species genome comparison

PubMed Central

Castrignanò, Tiziana; Canali, Alessandro; Grillo, Giorgio; Liuni, Sabino; Mignone, Flavio; Pesole, Graziano

2004-01-01

The identification and characterization of genome tracts that are highly conserved across species during evolution may contribute significantly to the functional annotation of whole-genome sequences. Indeed, such sequences are likely to correspond to known or unknown coding exons or regulatory motifs. Here, we present a web server implementing a previously developed algorithm that, by comparing user-submitted genome sequences, is able to identify statistically significant conserved blocks and assess their coding or noncoding nature through the measure of a coding potential score. The web tool, available at http://www.caspur.it/CSTminer/, is dynamically interconnected with the Ensembl genome resources and produces a graphical output showing a map of detected conserved sequences and annotated gene features. PMID:15215464

Evaluate Bull Trout Movements in the Tucannon and Lower Snake Rivers, 2004 Annual Report.

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

Faler, Michael P.; Mendel, Glen W.; Fulton, Carl

2005-11-01

We sampled and released 313 bull trout (Salvelinus confluentus) from the Tucannon River in 2004. Passive Integrated Transponder (PIT) tags were inserted in 231 of these individuals, and we detected existing PIT tags in an additional 44 bull trout. Twenty-five of these were also surgically implanted with radio-tags, and we monitored the movements of these fish throughout the year. Ten bull trout that were radio-tagged in 2003 were known to survive and carry their tags through the spring of 2004. One of these fish outmigrated into the Snake River in the fall, and remained undetected until February, when it's tagmore » was located near the confluence of Alkali Flat Creek and the Snake River. The remaining 9 fish spent the winter between Tucannon River miles 2.1 (Powers Road) and 36.0 (Tucannon Fish Hatchery). Seven of these fish retained their tags through the summer, and migrated to known spawning habitat prior to September 2004. During June and July, radio-tagged bull trout again exhibited a general upstream movement into the upper reaches of the Tucannon subbasin. As in past years, we observed some downstream movements of radio-tagged bull trout in mid to late September and throughout October, suggesting post spawning outmigrations. By late November and early December, radio tagged bull trout were relatively stationary, and were distributed from river mile 42 at Camp Wooten downstream to river mile 17, near the Highway 12 bridge. As in previous years, we did not collect data associated with objectives 2, 3, or 4 of this study, because we were unable to monitor migratory movement of radio-tagged bull trout into the vicinity of the hydropower dams on the main stem Snake River. Transmission tests of submerged Lotek model NTC-6-2 nano-tags in Lower Granite Pool showed that audible detection and individual tag identification was possible at depths of 20, 30, and 40 ft. We were able to maintain tag detection and code separation at all depths from both a boat and 200 ft. above water surface in a helicopter. However, we lost detection capability from 40 ft. water depth when we passed 700 ft. above the water surface in a helicopter. Two years of high tag loss, particularly after spawning, has prevented us from documenting fall and winter movements with an adequate sample of radio tagged bull trout. The high transmitter loss after spawning may be a reflection of high natural mortality for large, older age fish that we have been radio tagging to accommodate the longer life transmitters. Therefore, we reduced the size of the radio tags that we implanted, and delayed most of our collection and tagging of bull trout until after spawning. These changes are a new approach to try to maximize the number of radio tagged bull trout available post spawning to adequately document fall and winter movements and any use of the Snake River by bull trout from the Tucannon River.« less

Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis.

PubMed

Wang, Huping; Han, Wenyu; Takagi, Junichi; Cong, Yao

2018-05-11

Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag.

PubMed

Zhang, Mengjie; Martin, Jody L; Kumar, Mohit; Khairallah, Ramzi J; de Tombe, Pieter P

2015-11-01

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1' cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner. Copyright © 2015 the American Physiological Society.

Detection of receptor ligands by monitoring selective stabilization of a Renilla luciferase-tagged, constitutively active mutant, G-protein-coupled receptor

PubMed Central

Ramsay, Douglas; Bevan, Nicola; Rees, Stephen; Milligan, Graeme

2001-01-01

The wild-type β2-adrenoceptor and a constitutively active mutant of this receptor were C-terminally tagged with luciferase from the sea pansy Renilla reniformis. C-terminal addition of Renilla luciferase did not substantially alter the levels of expression of either form of the receptor, the elevated constitutive activity of the mutant β2-adrenoceptor nor the capacity of isoprenaline to elevate cyclic AMP levels in intact cells expressing these constructs. Treatment of cells expressing constitutively active mutant β2-adrenoceptor-Renilla luciferase with antagonist/inverse agonist ligands resulted in upregulation of levels of this polypeptide which could be monitored by the elevated luciferase activity. The pEC50 for ligand-induced luciferase upregulation and ligand affinity to bind the receptor were highly correlated. Similar upregulation could be observed following sustained treatment with agonist ligands. These effects were only observed at a constitutively active mutant of the β2-adrenoceptor. Co-expression of the wild-type β2-adrenoceptor C-terminally tagged with the luciferase from Photinus pyralis did not result in ligand-induced upregulation of the levels of activity of this luciferase. Co-expression of the constitutively active mutant β2-adrenoceptor-Renilla luciferase and an equivalent mutant of the α1b-adrenoceptor C-terminally tagged with green fluorescent protein allowed pharmacological selectivity of adrenoceptor antagonists to be demonstrated. This approach offers a sensitive and convenient means, which is amenable to high throughput analysis, to monitor ligand binding to a constitutively active mutant receptor. As no prior knowledge of receptor ligands is required this approach may be suitable to identify ligands at orphan G protein-coupled receptors. PMID:11350868

Rapid large-scale purification of myofilament proteins using a cleavable His6-tag

PubMed Central

Zhang, Mengjie; Martin, Jody L.; Kumar, Mohit; de Tombe, Pieter P.

2015-01-01

With the advent of high-throughput DNA sequencing, the number of identified cardiomyopathy-causing mutations has increased tremendously. As the majority of these mutations affect myofilament proteins, there is a need to understand their functional consequence on contraction. Permeabilized myofilament preparations coupled with protein exchange protocols are a common method for examining into contractile mechanics. However, producing large quantities of myofilament proteins can be time consuming and requires different approaches for each protein of interest. In the present study, we describe a unified automated method to produce troponin C, troponin T, and troponin I as well as myosin light chain 2 fused to a His6-tag followed by a tobacco etch virus (TEV) protease site. TEV protease has the advantage of a relaxed P1′ cleavage site specificity, allowing for no residues left after proteolysis and preservation of the native sequence of the protein of interest. After expression in Esherichia coli, cells were lysed by sonication in imidazole-containing buffer. The His6-tagged protein was then purified using a HisTrap nickel metal affinity column, and the His6-tag was removed by His6-TEV protease digestion for 4 h at 30°C. The protease was then removed using a HisTrap column, and complex assembly was performed via column-assisted sequential desalting. This mostly automated method allows for the purification of protein in 1 day and can be adapted to most soluble proteins. It has the advantage of greatly increasing yield while reducing the time and cost of purification. Therefore, production and purification of mutant proteins can be accelerated and functional data collected in a faster, less expensive manner. PMID:26386113

Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification.

PubMed

Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan

2017-03-24

In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc) 2 .4H 2 O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg -1 (protein/MnFe 2 O 4 @SiO 2 @Ni-Salen complex). Collectively, purification process with MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Tagging of Endogenous BK Channels with a Fluorogen-Activating Peptide Reveals β4-Mediated Control of Channel Clustering in Cerebellum

PubMed Central

Pratt, Christopher P.; Kuljis, Dika A.; Homanics, Gregg E.; He, Jianjun; Kolodieznyi, Dmytro; Dudem, Srikanth; Hollywood, Mark A.; Barth, Alison L.; Bruchez, Marcel P.

2017-01-01

BK channels are critical regulators of neuronal activity, controlling firing, neurotransmitter release, cerebellar function, and BK channel mutations have been linked to seizure disorders. Modulation of BK channel gating is well characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been less studied. Fluorogen-activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG)-based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. Cell-excluded MG dyes enable the selective tagging of surface protein and tracking through endocytic pathways. We used CRISPR to insert the FAP at the extracellular N-terminus of BKα in the first exon of its native locus, enabling regulation by the native promoter elements and tag incorporation into multiple splice isoforms. Motor coordination was found to be normal; however, BK channel expression seems to be reduced in some locations. Alternate start site selection or post-translational proteolytic processing resulted in incomplete FAP tagging of the BKα proteins in brain tissues. In Purkinje cell somata, FAP revealed BK channel clustering previously only observed by electron microscopy. Measurement of these clusters in β4+/- and β4-/- mice showed that puncta number and cluster fluorescence intensity on the soma are reduced in β4-/- knockout animals. This novel mouse line provides a versatile fluorescent platform for studying endogenous BK channels in living and fixed tissues. Future studies could apply this line to ex vivo neuronal cultures to study live-cell channel trafficking. PMID:29163049

Laboratory evidence for short and long-term damage to pink salmon incubating in oiled gravel

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

Heintz, R.; Rice, S.; Wiedmer, M.

1995-12-31

Pink salmon, incubating in gravel contaminated with crude oil, demonstrated immediate and delayed responses in the laboratory at doses consistent with the concentrations observed in oiled streams in Prince William Sound. The authors incubated pink salmon embryos in a simulated intertidal environment with gravel contaminated by oil from the Exxon Valdez. During the incubation and emergence periods the authors quantified dose-response curves for characters affected directly by the oil. After emergence, fish were coded wire tagged and released, or cultured in netpens. Delayed responses have been observed among the cultured fish, and further observations will be made when coded wiremore » tagged fish return in September 1995. The experiments have demonstrated that eggs need not contact oiled gravel to experience increased mortality, and doses as low as 17 ppb tPAH in water can have delayed effects on growth. A comparison of sediment tPAH concentrations from streams in Prince William Sound with these laboratory data suggests that many 1989 brood pink salmon were exposed to deleterious quantities of oil.« less

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

PubMed Central

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-01-01

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. PMID:28835375

Technologies for the marking of fishing gear to identify gear components entangled on marine animals and to reduce abandoned, lost or otherwise discarded fishing gear.

PubMed

He, Pingguo; Suuronen, Petri

2018-04-01

Fishing gears are marked to establish and inform origin, ownership and position. More recently, fishing gears are marked to aid in capacity control, reduce marine litter due to abandoned, lost or otherwise discarded fishing gear (ALDFG) and assist in its recovery, and to combat illegal, unreported and unregulated (IUU) fishing. Traditionally, physical marking, inscription, writing, color, shape, and tags have been used for ownership and capacity purposes. Buoys, lights, flags, and radar reflectors are used for marking of position. More recently, electronic devices have been installed on marker buoys to enable easier relocation of the gear by owner vessels. This paper reviews gear marking technologies with focus on coded wire tags, radio frequency identification tags, Automatic Identification Systems, advanced electronic buoys for pelagic longlines and fish aggregating devices, and re-location technology if the gear becomes lost. Copyright © 2018 Elsevier Ltd. All rights reserved.

Structural Characterization of a Therapeutic Anti-Methamphetamine Antibody Fragment: Oligomerization and Binding of Active Metabolites

PubMed Central

Gokulan, Kuppan; Varughese, Kottayil I.

2013-01-01

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, KD = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or “ecstasy”). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni2+. Two of the histidine residues of each C-terminal His-tag interact with Ni2+ in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy. PMID:24349338

Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins.

PubMed

Baumann, Pascal; Bluthardt, Nicolai; Renner, Sarah; Burghardt, Hannah; Osberghaus, Anna; Hubbuch, Jürgen

2015-04-20

Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag™ technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag™ was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced. Copyright © 2015 Elsevier B.V. All rights reserved.

Importance of the residue Asp 290 on chain length selectivity and catalytic efficiency of recombinant Staphylococcus simulans lipase expressed in E. coli.

PubMed

Sayari, Adel; Mosbah, Habib; Gargouri, Youssef

2007-05-01

In addition to their physiological importance, microbial lipases, like staphylococcal ones, are of considerable commercial interest for biotechnological applications such as detergents, food production, and pharmaceuticals and industrial synthesis of fine chemicals. The gene encoding the extracellular lipase of Staphylococcus simulans (SSL) was subcloned in the pET-14b expression vector and expressed in Esherichia coli BL21 (DE3). The wild-type SSL was expressed as amino terminal His6-tagged recombinant protein. One-step purification of the recombinant lipase was achieved with nickel metal affinity column. The purified His-tagged SSL (His6-SSL) is able to hydrolyse triacylglycerols without chain length selectivity. The major differences among lipases are reflected in their chemical specificity in the hydrolysis of peculiar ester bonds, and their respective capacity to hydrolyse substrates having different physico-chemical properties. It has been proposed, using homology alignment, that the region around the residue 290 of Staphylococcus hyicus lipase could be involved in the selection of the substrate. To evaluate the importance of this environment, the residue Asp290 of Staphylococcus simulans lipase was mutated to Ala using site-directed mutagenesis. The mutant expression plasmid was also overexpressed in Esherichia coli and purified with a nickel metal affinity column. The substitution of Asp290 by Ala was accompanied by a significant shift of the acyl-chain length specificity of the mutant towards short chain fatty acid esters. Kinetic studies of wild-type SSL and its mutant D290A were carried out, and show essentially that the catalytic efficiency (k cat /K M ) of the mutant was affected. Our results confirmed that Asp290 is important for the chain length selectivity and catalytic efficiency of Staphylococcus simulans lipase.

Structural characterization of a therapeutic anti-methamphetamine antibody fragment: oligomerization and binding of active metabolites.

PubMed

Peterson, Eric C; Celikel, Reha; Gokulan, Kuppan; Varughese, Kottayil I

2013-01-01

Vaccines and monoclonal antibodies (mAb) for treatment of (+)-methamphetamine (METH) abuse are in late stage preclinical and early clinical trial phases, respectively. These immunotherapies work as pharmacokinetic antagonists, sequestering METH and its metabolites away from sites of action in the brain and reduce the rewarding and toxic effects of the drug. A key aspect of these immunotherapy strategies is the understanding of the subtle molecular interactions important for generating antibodies with high affinity and specificity for METH. We previously determined crystal structures of a high affinity anti-METH therapeutic single chain antibody fragment (scFv6H4, K(D) = 10 nM) in complex with METH and the (+) stereoisomer of 3,4-methylenedioxymethamphetamine (MDMA, or "ecstasy"). Here we report the crystal structure of scFv6H4 in homo-trimeric unbound (apo) form (2.60Å), as well as monomeric forms in complex with two active metabolites; (+)-amphetamine (AMP, 2.38Å) and (+)-4-hydroxy methamphetamine (p-OH-METH, 2.33Å). The apo structure forms a trimer in the crystal lattice and it results in the formation of an intermolecular composite beta-sheet with a three-fold symmetry. We were also able to structurally characterize the coordination of the His-tags with Ni(2+). Two of the histidine residues of each C-terminal His-tag interact with Ni(2+) in an octahedral geometry. In the apo state the CDR loops of scFv6H4 form an open conformation of the binding pocket. Upon ligand binding, the CDR loops adopt a closed formation, encasing the drug almost completely. The structural information reported here elucidates key molecular interactions important in anti-methamphetamine abuse immunotherapy.

Expression and purification of mouse peptide ESP4 in Escherichia coli.

PubMed

Hirakane, Makoto; Taniguchi, Masahiro; Yoshinaga, Sosuke; Misumi, Shogo; Terasawa, Hiroaki

2014-04-01

Pheromones are species-specific chemical signals that regulate a wide range of social and sexual behaviors in many animals. In mice, the male-specific peptide ESP1 (exocrine gland-secreting peptide 1) is secreted into tear fluids and enhances female sexual receptive behavior. ESP1 belongs to the ESP family, a multigene family with 38 genes in mice. ESP1 shares the highest homology with ESP4. ESP1 is expressed in the extraorbital lacrimal gland, whereas ESP4 is expressed in some exocrine glands. Thus, ESP4 is expected to have a function that has not been elucidated yet. Large amounts of the purified ESP4 protein are required for structural and biochemical studies. Here we present an expression and purification scheme for the recombinant ESP4 protein. The N-terminally histidine-tagged ESP4 fusion protein was expressed in Escherichia coli as inclusion bodies, which were solubilized and purified by nickel affinity chromatography. The histidine tag was cleaved with thrombin and removed by a second nickel affinity chromatography step. The ESP4 protein was isolated with high purity by reversed-phase chromatography. For NMR analyses, we prepared a stable isotope-labeled ESP4 protein. Three repeated freeze-drying steps after the reversed-phase chromatography were required, to remove a volatile contaminating compound and to obtain an NMR spectrum with a homogeneous line shape. AMS-modification and far-UV CD spectroscopic analyses suggested that ESP4 has an intramolecular disulfide bridge and a helical structure, respectively. The present study provides a powerful tool for structural and biochemical studies of ESP4, leading toward the elucidation of the roles of the ESP family members. Copyright © 2014 Elsevier Inc. All rights reserved.

Platform-independent and label-free quantitation of proteomic data using MS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation.

PubMed

Schilling, Birgit; Rardin, Matthew J; MacLean, Brendan X; Zawadzka, Anna M; Frewen, Barbara E; Cusack, Michael P; Sorensen, Dylan J; Bereman, Michael S; Jing, Enxuan; Wu, Christine C; Verdin, Eric; Kahn, C Ronald; Maccoss, Michael J; Gibson, Bradford W

2012-05-01

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.

Platform-independent and Label-free Quantitation of Proteomic Data Using MS1 Extracted Ion Chromatograms in Skyline

PubMed Central

Schilling, Birgit; Rardin, Matthew J.; MacLean, Brendan X.; Zawadzka, Anna M.; Frewen, Barbara E.; Cusack, Michael P.; Sorensen, Dylan J.; Bereman, Michael S.; Jing, Enxuan; Wu, Christine C.; Verdin, Eric; Kahn, C. Ronald; MacCoss, Michael J.; Gibson, Bradford W.

2012-01-01

Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models. PMID:22454539

Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

PubMed Central

Kaur, Jasvir; Reinhardt, Dieter P.

2012-01-01

Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

The optimal code searching method with an improved criterion of coded exposure for remote sensing image restoration

NASA Astrophysics Data System (ADS)

He, Lirong; Cui, Guangmang; Feng, Huajun; Xu, Zhihai; Li, Qi; Chen, Yueting

2015-03-01

Coded exposure photography makes the motion de-blurring a well-posed problem. The integration pattern of light is modulated using the method of coded exposure by opening and closing the shutter within the exposure time, changing the traditional shutter frequency spectrum into a wider frequency band in order to preserve more image information in frequency domain. The searching method of optimal code is significant for coded exposure. In this paper, an improved criterion of the optimal code searching is proposed by analyzing relationship between code length and the number of ones in the code, considering the noise effect on code selection with the affine noise model. Then the optimal code is obtained utilizing the method of genetic searching algorithm based on the proposed selection criterion. Experimental results show that the time consuming of searching optimal code decreases with the presented method. The restoration image is obtained with better subjective experience and superior objective evaluation values.

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments.

PubMed

Cannataro, Mario; Cuda, Giovanni; Gaspari, Marco; Greco, Sergio; Tradigo, Giuseppe; Veltri, Pierangelo

2007-07-15

Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L) pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein identification process and, consequently, on the amount of potentially critical information in clinical studies. The EIPeptiDi tool is available at http://bioingegneria.unicz.it/~veltri/projects/eipeptidi/ with a demo data set. EIPeptiDi significantly increases the number of peptides identified and quantified in analyzed samples, thus reducing the number of unassigned H/L pairs and allowing a better comparative analysis of sample data sets.

Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.

PubMed

Baldwin, M A; Medzihradszky, K F; Lock, C M; Fisher, B; Settineri, T A; Burlingame, A L

2001-04-15

The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.

Feasibility study for combination of field-flow fractionation (FFF)-based separation of size-coded particle probes with amplified surface enhanced Raman scattering (SERS) tagging for simultaneous detection of multiple miRNAs.

PubMed

Shin, Kayeong; Choi, Jaeyeong; Kim, Yeoju; Lee, Yoonjeong; Kim, Joohoon; Lee, Seungho; Chung, Hoeil

2018-06-29

We propose a new analytical scheme in which field-flow fractionation (FFF)-based separation of target-specific polystyrene (PS) particle probes of different sizes are incorporated with amplified surface-enhanced Raman scattering (SERS) tagging for the simultaneous and sensitive detection of multiple microRNAs (miRNAs). For multiplexed detection, PS particles of three different diameters (15, 10, 5 μm) were used for the size-coding, and a probe single stranded DNA (ssDNA) complementary to a target miRNA was conjugated on an intended PS particle. After binding of a target miRNA on PS probe, polyadenylation reaction was executed to generate a long tail composed of adenine (A) serving as a binding site to thymine (T) conjugated Au nanoparticles (T-AuNPs) to increase SERS intensity. The three size-coded PS probes bound with T-AuNPs were then separated in a FFF channel. With the observation of extinction-based fractograms, separation of three size-coded PS probes was clearly confirmed, thereby enabling of measuring three miRNAs simultaneously. Raman intensities of FFF fractions collected at the peak maximum of 15, 10 and 5 μm PS probes varied fairy quantitatively with the change of miRNA concentrations, and the reproducibility of measurement was acceptable. The proposed method is potentially useful for simultaneous detection of multiple miRNAs with high sensitivity. Copyright © 2018 Elsevier B.V. All rights reserved.

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

PubMed Central

Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

2014-01-01

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

PubMed

Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M; Climent, Víctor; Sanz, Jesús M

2014-01-01

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

Enhancing Chemical Inventory Management in Laboratory through a Mobile-Based QR Code Tag

NASA Astrophysics Data System (ADS)

Shukran, M. A. M.; Ishak, M. S.; Abdullah, M. N.

2017-08-01

The demand for a greater inventory management system which can provide a lot of useful information from a single scan has made laboratory inventory management using barcode technology more difficult. Since the barcode technology lacks the ability to overcome the problem and is not capable of providing information needed to manage the chemicals in the laboratory, thus employing a QR code technology is the best solution. In this research, the main idea is to develop a standalone application running with its own database that is periodically synchronized with the inventory software hosted by the computer and connected to a specialized network as well. The first process required to establish this centralized system is to determine all inventory available in the chemical laboratory by referring to the documented data in order to develop the database. Several customization and enhancement were made to the open source QR code technology to ensure the developed application is dedicated for its main purposes. As the end of the research, it was proven that the system is able to track the position of all inventory and showing real time information about the scanned chemical labels. This paper intends to give an overview about the QR tag inventory system that was developed and its implementation at the National Defence University of Malaysia’s (NDUM) chemical laboratory.

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein–protein interactions directly from DNA

PubMed Central

Layton, Curtis J; Hellinga, Homme W

2011-01-01

Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein–protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody–affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed. PMID:21674663

Multiplexed Affinity-Based Separation of Proteins and Cells Using Inertial Microfluidics.

PubMed

Sarkar, Aniruddh; Hou, Han Wei; Mahan, Alison E; Han, Jongyoon; Alter, Galit

2016-03-30

Isolation of low abundance proteins or rare cells from complex mixtures, such as blood, is required for many diagnostic, therapeutic and research applications. Current affinity-based protein or cell separation methods use binary 'bind-elute' separations and are inefficient when applied to the isolation of multiple low-abundance proteins or cell types. We present a method for rapid and multiplexed, yet inexpensive, affinity-based isolation of both proteins and cells, using a size-coded mixture of multiple affinity-capture microbeads and an inertial microfluidic particle sorter device. In a single binding step, different targets-cells or proteins-bind to beads of different sizes, which are then sorted by flowing them through a spiral microfluidic channel. This technique performs continuous-flow, high throughput affinity-separation of milligram-scale protein samples or millions of cells in minutes after binding. We demonstrate the simultaneous isolation of multiple antibodies from serum and multiple cell types from peripheral blood mononuclear cells or whole blood. We use the technique to isolate low abundance antibodies specific to different HIV antigens and rare HIV-specific cells from blood obtained from HIV+ patients.

PolyCheck: Dynamic Verification of Iteration Space Transformations on Affine Programs

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

Bao, Wenlei; Krishnamoorthy, Sriram; Pouchet, Louis-noel

2016-01-11

High-level compiler transformations, especially loop transformations, are widely recognized as critical optimizations to restructure programs to improve data locality and expose parallelism. Guaranteeing the correctness of program transformations is essential, and to date three main approaches have been developed: proof of equivalence of affine programs, matching the execution traces of programs, and checking bit-by-bit equivalence of the outputs of the programs. Each technique suffers from limitations in either the kind of transformations supported, space complexity, or the sensitivity to the testing dataset. In this paper, we take a novel approach addressing all three limitations to provide an automatic bug checkermore » to verify any iteration reordering transformations on affine programs, including non-affine transformations, with space consumption proportional to the original program data, and robust to arbitrary datasets of a given size. We achieve this by exploiting the structure of affine program control- and data-flow to generate at compile-time lightweight checker code to be executed within the transformed program. Experimental results assess the correctness and effectiveness of our method, and its increased coverage over previous approaches.« less

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Hwang, Jung-Shan; Yamada, Kazunori; Honda, Ayae; Nakade, Kohji; Ishihama, Akira

2000-01-01

Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni2+-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. PMID:10756019

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.

Functional study of miR-27a in human hepatic stellate cells by proteomic analysis: comprehensive view and a role in myogenic tans-differentiation.

PubMed

Ji, Yuhua; Zhang, Jinsheng; Wang, Wenwen; Ji, Juling

2014-01-01

We previous reported that miR-27a regulates lipid metabolism and cell proliferation during hepatic stellate cells (HSCs) activation. To further explore the biological function and underlying mechanisms of miR-27a in HSCs, global protein expression affected by overexpression of miR-27a in HSCs was analyzed by a cleavable isotope-coded affinity tags (cICAT) based comparative proteomic approach. In the present study, 1267 non-redundant proteins were identified with unique accession numbers (score ≥1.3, i.e. confidence ≥95%), among which 1171 were quantified and 149 proteins (12.72%) were differentially expressed with a differential expression ratio of 1.5. We found that up-regulated proteins by miR-27a mainly participate in cell proliferation and myogenesis, while down-regulated proteins were the key enzymes involved in de novo lipid synthesis. The expression of a group of six miR-27a regulated proteins was validated and the function of one miR-27a regulated protein was further validated. The results not only delineated the underlying mechanism of miR-27a in modulating fat metabolism and cell proliferation, but also revealed a novel role of miR-27a in promoting myogenic tans-differentiation during HSCs activation. This study also exemplified proteomics strategy as a powerful tool for the functional study of miRNA.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies.

PubMed Central

Chow, L P; Liu, S L; Yu, C J; Liao, H K; Tsai, J J; Tang, T K

2000-01-01

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein. PMID:10677362

Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.

PubMed

Zhu, Mengmeng; Zhu, Ning; Song, Wen-yuan; Harmon, Alice C; Assmann, Sarah M; Chen, Sixue

2014-05-01

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox-sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard-cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis and isotope-coded affinity tagging. In total, 65 and 118 potential redox-responsive proteins were identified in ABA- and MeJA-treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of 'energy', 'stress and defense' and 'metabolism'. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA- and MeJA-treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non-fermenting 1-related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox-regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water-use efficiency and abiotic stress tolerance in plants.

PubMed

Moin, Mazahar; Bakshi, Achala; Saha, Anusree; Udaya Kumar, M; Reddy, Attipalli R; Rao, K V; Siddiq, E A; Kirti, P B

2016-11-01

We have generated 3900 enhancer-based activation-tagged plants, in addition to 1030 stable Dissociator-enhancer plants in a widely cultivated indica rice variety, BPT-5204. Of them, 3000 were screened for water-use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield-related attributes under water-limiting conditions that identified 200 activation-tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ 13 C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up-regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating-tagging. The high up-regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis-regulatory elements in their promoter regions along with the high up-regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance. © 2016 John Wiley & Sons Ltd.

Swarmathon 2017

NASA Image and Video Library

2017-04-20

In the Swarmathon competition at the Kennedy Space Center Visitor Complex, students were asked to develop computer code for the small robots, programming them to look for "resources" in the form of cubes with AprilTags, similar to barcodes. Teams developed search algorithms for the Swarmies to operate autonomously, communicating and interacting as a collective swarm similar to ants foraging for food.

50 CFR 23.74 - How can I trade internationally in personal sport-hunted trophies?

Code of Federal Regulations, 2014 CFR

2014-10-01

... certificate must contain all the information that is given on the tag. (2) African elephant (Loxodonta... sport-hunted trophies of African elephant. The trophy ivory must be legibly marked by means of punch... corresponding two-letter ISO country code; the last two digits of the year in which the elephant was harvested...

50 CFR Table 1b to Part 679 - Discard and Disposition Codes

Code of Federal Regulations, 2010 CFR

2010-10-01

.... 63 Deadloss (crab only). 79 Halibut retained for future sale (Halibut only). 87 Overage (Specify overage type in comment). 62 Tagged Fish (Exempt from IFQ). 64 Whole fish/bait, not sold. Used as bait on board vessel. 92 Whole fish/bait, sold. 61 Whole fish/discard at sea.Whole groundfish and prohibited...

50 CFR Table 1b to Part 679 - Discard and Disposition Codes1

Code of Federal Regulations, 2014 CFR

2014-10-01

... Confiscation or seized 63 Deadloss (crab only) 79 Overage 62 Retained for future sale 87 Tagged IFQ Fish (Exempt from debit) 64 Whole fish/bait, not sold. Used as bait onboard vessel 92 Whole fish/bait, sold 61 Whole fish/discard at sea. Whole groundfish and prohibited species discarded by catcher vessels, catcher...

50 CFR Table 1b to Part 679 - Discard and Disposition Codes1

Code of Federal Regulations, 2012 CFR

2012-10-01

... Confiscation or seized 63 Deadloss (crab only) 79 Overage 62 Retained for future sale 87 Tagged IFQ Fish (Exempt from debit) 64 Whole fish/bait, not sold. Used as bait onboard vessel 92 Whole fish/bait, sold 61 Whole fish/discard at sea. Whole groundfish and prohibited species discarded by catcher vessels, catcher...

50 CFR Table 1b to Part 679 - Discard and Disposition Codes1

Code of Federal Regulations, 2011 CFR

2011-10-01

... Confiscation or seized 63 Deadloss (crab only) 79 Overage 62 Retained for future sale 87 Tagged IFQ Fish (Exempt from debit) 64 Whole fish/bait, not sold. Used as bait onboard vessel 92 Whole fish/bait, sold 61 Whole fish/discard at sea. Whole groundfish and prohibited species discarded by catcher vessels, catcher...

50 CFR Table 1b to Part 679 - Discard and Disposition Codes1

Code of Federal Regulations, 2013 CFR

2013-10-01

... Confiscation or seized 63 Deadloss (crab only) 79 Overage 62 Retained for future sale 87 Tagged IFQ Fish (Exempt from debit) 64 Whole fish/bait, not sold. Used as bait onboard vessel 92 Whole fish/bait, sold 61 Whole fish/discard at sea. Whole groundfish and prohibited species discarded by catcher vessels, catcher...

48 CFR 252.211-7007 - Reporting of Government-Furnished Equipment in the DoD Item Unique Identification (IUID) Registry.

Code of Federal Regulations, 2011 CFR

2011-10-01

... Organization/International Electrotechnical Commission (ISO/IEC) Standard 16022: Information Technology... for sale, and does not ordinarily lose its identity or become a component part of another article when... code (for items too small to individually tag or mark). (ii) Contents (the type of information recorded...

Swarmathon 2017

NASA Image and Video Library

2017-04-20

This close-up shows Swarmie robots that were programmed with computer code by college and university students. During the Swarmathon competition at the Kennedy Space Center Visitor Complex, the small robots looked for "resources" in the form of cubes with AprilTags, similar to barcodes. Similar robots could help find resources when astronauts explore distant locations, such as the moon or Mars.

Tumor-targeting domains for chimeric antigen receptor T cells.

PubMed

Bezverbnaya, Ksenia; Mathews, Ashish; Sidhu, Jesse; Helsen, Christopher W; Bramson, Jonathan L

2017-01-01

Immunotherapy with chimeric antigen receptor (CAR) T cells has been advancing steadily in clinical trials. Since the ability of engineered T cells to recognize intended tumor-associated targets is crucial for the therapeutic success, antigen-binding domains play an important role in shaping T-cell responses. Single-chain antibody and T-cell receptor fragments, natural ligands, repeat proteins, combinations of the above and universal tag-specific domains have all been used in the antigen-binding moiety of chimeric receptors. Here we outline the advantages and disadvantages of different domains, discuss the concepts of affinity and specificity, and highlight the recent progress of each targeting strategy.

Electrochemical genosensing of Salmonella, Listeria and Escherichia coli on silica magnetic particles.

PubMed

Liébana, Susana; Brandão, Delfina; Cortés, Pilar; Campoy, Susana; Alegret, Salvador; Pividori, María Isabel

2016-01-21

A magneto-genosensing approach for the detection of the three most common pathogenic bacteria in food safety, such as Salmonella, Listeria and Escherichia coli is presented. The methodology is based on the detection of the tagged amplified DNA obtained by single-tagging PCR with a set of specific primers for each pathogen, followed by electrochemical magneto-genosensing on silica magnetic particles. A set of primers were selected for the amplification of the invA (278 bp), prfA (217 bp) and eaeA (151 bp) being one of the primers for each set tagged with fluorescein, biotin and digoxigenin coding for Salmonella enterica, Listeria monocytogenes and E. coli, respectively. The single-tagged amplicons were then immobilized on silica MPs based on the nucleic acid-binding properties of silica particles in the presence of the chaotropic agent as guanidinium thiocyanate. The assessment of the silica MPs as a platform for electrochemical magneto-genosensing is described, including the main parameters to selectively attach longer dsDNA fragments instead of shorter ssDNA primers based on their negative charge density of the sugar-phosphate backbone. This approach resulted to be a promising detection tool with sensing features of rapidity and sensitivity very suitable to be implemented on DNA biosensors and microfluidic platforms. Copyright © 2015 Elsevier B.V. All rights reserved.

Swarmathon 2017

NASA Image and Video Library

2017-04-19

In the Swarmathon competition at the Kennedy Space Center Visitor Complex, students were asked to develop computer code for the small robots, programming them to look for "resources" in the form of AprilTag cubes, similar to barcodes. Teams developed search algorithms for the Swarmies to operate autonomously, communicating and interacting as a collective swarm similar to ants foraging for food. In the spaceport's second annual Swarmathon, 20 teams representing 22 minority serving universities and community colleges were invited to develop software code to operate these innovative robots known as "Swarmies" to help find resources when astronauts explore distant locations, such as the moon or Mars.

Swarmathon 2018

NASA Image and Video Library

2018-04-18

In the Swarmathon competition at the Kennedy Space Center Visitor Complex, students were asked to develop computer code for the small robots, programming them to look for "resources" in the form of AprilTag cubes, similar to barcodes. Teams developed search algorithms for the Swarmies to operate autonomously, communicating and interacting as a collective swarm similar to ants foraging for food. In the spaceport's third annual Swarmathon, 23 teams represented 24 minority serving universities and community colleges were invited to develop software code to operate these innovative robots known as "Swarmies" to help find resources when astronauts explore distant locations, such as the Moon or Mars.

Swarmathon 2018

NASA Image and Video Library

2018-04-17

In the Swarmathon competition at the Kennedy Space Center Visitor Complex, students were asked to develop computer code for the small robots, programming them to look for "resources" in the form of AprilTag cubes, similar to barcodes. Teams developed search algorithms for the Swarmies to operate autonomously, communicating and interacting as a collective swarm similar to ants foraging for food. In the spaceport's third annual Swarmathon, 23 teams represented 24 minority serving universities and community colleges were invited to develop software code to operate these innovative robots known as "Swarmies" to help find resources when astronauts explore distant locations, such as the Moon or Mars.

Movements of Atlantic Sturgeon of the Gulf of Maine inside and outside the geographically defined Distinct Population Segment

USGS Publications Warehouse

Wippelhauser, Gail S.; Sulikowski, James; Zydlewski, Gayle B.; Altenritter, Megan; Kieffer, Micah; Kinnison, Michael T.

2017-01-01

Identification of potential critical habitat, seasonal distributions, and movements within and between river systems is important for protecting the Gulf of Maine (GOM) Distinct Population Segment of Atlantic Sturgeon. To accomplish these objectives, we captured Atlantic Sturgeon in four GOM rivers (Penobscot, Kennebec system, Saco, and Merrimack), and tagged 144 (83.3–217.4 cm TL) internally with uniquely coded acoustic transmitters. Tagged fish were detected between 2006 to 2014 by primary receiver arrays deployed in the four GOM rivers or opportunistically on a secondary group of receivers deployed within the GOM and along the continental shelf. Atlantic Sturgeon tagged in the four rivers were documented at three spawning areas in the Kennebec system in June and July, including one that became accessible in 1999 when the Edwards Dam was removed. After being tagged, the majority (74%) of Atlantic sturgeon were detected in the estuaries of the four GOM rivers, primarily from May through October. Tagged fish spent most of their time in saline water in the Saco River and Merrimack River, moved into brackish water in the Penobscot River, and were found in saline, brackish, and fresh water in the Kennebec system. Approximately 70% of the tagged fish were detected in GOM coastal waters, and aggregated in the Bay of Fundy (May–January), offshore of the Penobscot River (September-February and May), offshore of the Kennebec River (September–February), in Saco Bay and the Scarborough River (July–November), and along the eastern Massachusetts coast between Cape Ann and Cape Cod (April–February). Nine tagged Atlantic sturgeon (7%) left the GOM, three of which moved as far north as Halifax in Canada and six moved as far south as the James River in Virginia. Information from this study will be used to make recommendations to avoid, reduce or mitigate the impacts of in-water projects and on Atlantic sturgeon.

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

PubMed

Kamoun, Jannet; Schué, Mathieu; Messaoud, Wala; Baignol, Justine; Point, Vanessa; Mateos-Diaz, Eduardo; Mansuelle, Pascal; Gargouri, Youssef; Parsiegla, Goetz; Cavalier, Jean-François; Carrière, Frédéric; Aloulou, Ahmed

2015-02-01

Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica. Copyright © 2014 Elsevier B.V. All rights reserved.

The use of tags and tag clouds to discern credible content in online health message forums.

PubMed

O'Grady, Laura; Wathen, C Nadine; Charnaw-Burger, Jill; Betel, Lisa; Shachak, Aviv; Luke, Robert; Hockema, Stephen; Jadad, Alejandro R

2012-01-01

Web sites with health-oriented content are potentially harmful if inaccurate or inappropriate medical information is used to make health-related decisions. Checklists, rating systems and guidelines have been developed to help people determine what is credible, but recent Internet technologies emphasize applications that are collaborative in nature, including tags and tag clouds, where site users 'tag' or label online content, each using their own labelling system. Concepts such as the date, reference, author, testimonial and quotations are considered predictors of credible content. An understanding of these descriptive tools, how they relate to the depiction of credibility and how this relates to overall efforts to label data in relation to the semantic web has yet to emerge. This study investigates how structured (pre-determined) and unstructured (user-generated) tags and tag clouds with a multiple word search feature are used by participants to assess credibility of messages posted in online message forums. The targeted respondents were those using web sites message forums for disease self-management. We also explored the relevancy of our findings to the labelling or indexing of data in the context of the semantic web. Diabetes was chosen as the content area in this study, since (a) this is a condition with increasing prevalence and (b) diabetics have been shown to actively use the Internet to manage their condition. From January to March 2010 participants were recruited using purposive sampling techniques. A screening instrument was used to determine eligibility. The study consisted of a demographic and computer usage survey, a series of usability tests and an interview. We tested participants (N=22) on two scenarios, each involving tasks that assessed their ability to tag content and search using a tag cloud that included six structured credibility terms (statistics, date, reference, author, testimonial and quotations). MORAE Usability software (version 3.1) was employed to record participants' use of the study environment. The surveys were analyzed using SPSS version 17. Interviews with participants were transcribed, coded and analyzed using thematic text analysis with the aid of NVivo8. Most participants had experience with Internet resources. However, less than one quarter of this sample had seen or used tags or a tag clouds. The ways in which participants used tags to label the content posted in the message forums varied. Some participants were tagging the information for their own subsequent use, whereas others viewed this process from the perspective of others: they tagged the content in ways that they thought other users would find beneficial. Many participants did not use the structured credibility tags when asked to search for credible content. The interviews corroborated these findings by confirming participants were not considering credibility foremost when tagging. Many participants in this study focused on assessing whether the information was relevant to their current circumstances, after which they would proceed to determine its credibility by corroborating with other sources. The use of structured tags to label information may not be a useful way to encourage the use of tagging, or to indicate credibility in this context. Current applications used in the semantic web automate this process. Therefore it may be useful to engage consumers of online content, in particular health-related content, to be more directly involved in the annotation of this content. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

PubMed

Muro, Eleonora; Hoang, Thang Q; Jobart-Malfait, Aude; Hernandez-Verdun, Danièle

2008-05-01

The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.

Frameshifting in the p6 cDNA phage display system.

PubMed

Govarts, Cindy; Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2010-12-20

Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.

Heterofunctional Magnetic Metal-Chelate-Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity.

PubMed

Tural, Servet; Tural, Bilsen; Demir, Ayhan S

2015-09-01

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly-His-tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor-made magnetic chelate-epoxy supports. In order to selectively adsorb and then covalently immobilize the poly-His-tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co(2+)-chelate groups (38 µmol Co(2+)/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine-tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free-enzyme-catalyzed reaction. The enantiomeric excess (ee) of (R)-benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles. © 2015 Wiley Periodicals, Inc.

Affinity purification–mass spectrometry and network analysis to understand protein-protein interactions

PubMed Central

Morris, John H; Knudsen, Giselle M; Verschueren, Erik; Johnson, Jeffrey R; Cimermancic, Peter; Greninger, Alexander L; Pico, Alexander R

2015-01-01

By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification–mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography–MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one’s familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2–4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one’s data and find the right questions. PMID:25275790

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

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

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

PubMed

Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

2016-12-01

The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

Direct and selective immobilization of proteins by means of an inorganic material-binding peptide: discussion on functionalization in the elongation to material-binding peptide.

PubMed

Yokoo, Nozomi; Togashi, Takanari; Umetsu, Mitsuo; Tsumoto, Kouhei; Hattori, Takamitsu; Nakanishi, Takeshi; Ohara, Satoshi; Takami, Seiichi; Naka, Takashi; Abe, Hiroya; Kumagai, Izumi; Adschiri, Tadafumi

2010-01-14

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.

Efficient fabrication of high-capacity immobilized metal ion affinity chromatographic media: The role of the dextran-grafting process and its manipulation.

PubMed

Zhao, Lan; Zhang, Jingfei; Huang, Yongdong; Li, Qiang; Zhang, Rongyue; Zhu, Kai; Suo, Jia; Su, Zhiguo; Zhang, Zhigang; Ma, Guanghui

2016-03-01

Novel high-capacity Ni(2+) immobilized metal ion affinity chromatographic media were prepared through the dextran-grafting process. Dextran was grafted to an allyl-activated agarose-based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran-grafted agarose microspheres and partly dextran-grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran-grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran-grafted microspheres, while just distributed around the outside of the partly dextran-grafted ones. Flow hydrodynamic properties were improved greatly after the dextran-grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran-grafting process, and partly dextran-grafted Ni(2+) chelating medium had a maximum binding capacity for His-tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high-efficiency and large-scale protein purification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Specific DNA binding of the two chicken Deformed family homeodomain proteins, Chox-1.4 and Chox-a.

PubMed Central

Sasaki, H; Yokoyama, E; Kuroiwa, A

1990-01-01

The cDNA clones encoding two chicken Deformed (Dfd) family homeobox containing genes Chox-1.4 and Chox-a were isolated. Comparison of their amino acid sequences with another chicken Dfd family homeodomain protein and with those of mouse homologues revealed that strong homologies are located in the amino terminal regions and around the homeodomains. Although homologies in other regions were relatively low, some short conserved sequences were also identified. E. coli-made full length proteins were purified and used for the production of specific antibodies and for DNA binding studies. The binding profiles of these proteins to the 5'-leader and 5'-upstream sequences of Chox-1.4 and Chox-a coding regions were analyzed by immunoprecipitation and DNase I footprint assays. These two Chox proteins bound to the same sites in the 5'-flanking sequences of their coding regions with various affinities and their binding affinities to each site were nearly the same. The consensus sequences of the high and low affinity binding sites were TAATGA(C/G) and CTAATTTT, respectively. A clustered binding site was identified in the 5'-upstream of the Chox-a gene, suggesting that this clustered binding site works as a cis-regulatory element for auto- and/or cross-regulation of Chox-a gene expression. Images PMID:1970866