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Sample records for affinity purification-tagged proteins

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

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    Colin P De Souza

    Full Text Available 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

  2. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants

    OpenAIRE

    Frank eSainsbury; Philippe V. Jutras; Juan eVorster; Marie-Claire eGoulet; Dominique eMichaud

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizi...

  3. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants

    Directory of Open Access Journals (Sweden)

    Frank eSainsbury

    2016-02-01

    Full Text Available The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to rapidly purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues.

  4. Affinity Purification of a Recombinant Protein Expressed as a Fusion with the Maltose-Binding Protein (MBP) Tag

    Science.gov (United States)

    Duong-Ly, Krisna C.; Gabelli, Sandra B.

    2015-01-01

    Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors as a first step in the purification of a recombinant protein fused with MBP, using fast protein liquid chromatography (FPLC). Cells should have been thawed, resuspended in binding buffer, and lysed by sonication or microfluidization before mixing with the amylose resin or loading on the column. Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment. PMID:26096500

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

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    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.

  6. Spectral affinity in protein networks

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    Teng Shang-Hua

    2009-11-01

    Full Text Available Abstract Background Protein-protein interaction (PPI networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise than simpler methods based on edge density and shortest path length. Results We develop a novel affinity measure for pairs of proteins in PPI networks, which uses personalized PageRank, a random walk based method used in context-sensitive search on the Web. Our measure of closeness, which we call PageRank Affinity, is proportional to the number of times the smaller-degree protein is visited in a random walk that restarts at the larger-degree protein. PageRank considers paths of all lengths in a network, therefore PageRank Affinity is a precise measure that is robust to noise in the data. PageRank Affinity is also provably related to cluster co-membership, making it a meaningful measure. In our experiments on protein networks we find that our measure is better at predicting co-complex membership and finding functionally related proteins than other commonly used measures of closeness. Moreover, our experiments indicate that PageRank Affinity is very resilient to noise in the network. In addition, based on our method we build a tool that quickly finds nodes closest to a queried protein in any protein network, and easily scales to much larger biological networks. Conclusion We define a meaningful way to assess the closeness of two proteins in a PPI network, and show that our closeness measure is more biologically significant than other commonly used methods. We also develop a tool, accessible at http://xialab.bu.edu/resources/pnns, that allows the user to

  7. Protein Complex Purification by Affinity Capture.

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    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  8. Protein isolation using affinity chromatography

    OpenAIRE

    Besselink, T.

    2012-01-01

    Many product or even waste streams in the food industry contain components that may have potential for e.g. functional foods. These streams are typically large in volume and the components of interest are only present at low concentrations. A robust and highly selective separation process should be developed for efficient isolation of the components. Affinity chromatography is such a selective method. Ligands immobilized to a stationary phase (e.g., a resin or membrane) are used to bind the c...

  9. Spectral affinity in protein networks

    OpenAIRE

    Teng Shang-Hua; Voevodski Konstantin; Xia Yu

    2009-01-01

    Abstract Background Protein-protein interaction (PPI) networks enable us to better understand the functional organization of the proteome. We can learn a lot about a particular protein by querying its neighborhood in a PPI network to find proteins with similar function. A spectral approach that considers random walks between nodes of interest is particularly useful in evaluating closeness in PPI networks. Spectral measures of closeness are more robust to noise in the data and are more precise...

  10. Optimized Affinity Capture of Yeast Protein Complexes.

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    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  11. Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum.

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    Kollmar, Martin

    2006-08-15

    The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species. PMID:16516959

  12. Detection of protein-protein interactions using tandem affinity purification.

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    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  13. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

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    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  14. Affinity Purification of Protein Complexes Using TAP Tags

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    Gerace, Erica; Moazed, Danesh

    2016-01-01

    This protocol is used for the isolation and analysis of protein complexes using the tandem affinity purification (TAP) tag system. The protocol describes the purification of a protein fused to a TAP tag comprised of two protein A domains and the calmodulin binding peptide separated by a TEV cleavage site. This is a powerful technique for rapid purification of protein complexes and the analysis of their stoichiometric composition, posttranslational modifications, structure, and functional activities. PMID:26096502

  15. Affitins for protein purification by affinity magnetic fishing.

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    Fernandes, Cláudia S M; Dos Santos, Raquel; Ottengy, Stella; Viecinski, Aline Canani; Béhar, Ghislaine; Mouratou, Barbara; Pecorari, Frédéric; Roque, A Cecília A

    2016-07-29

    Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing. PMID:27342136

  16. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

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    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  17. The Protein-Protein Interface Evolution Acts in a Similar Way to Antibody Affinity Maturation*

    OpenAIRE

    Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

    2009-01-01

    Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniq...

  18. Expression and affinity purification of recombinant proteins from plants

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    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).

  19. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

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    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  20. A NOVEL METAL CHELATE AFFINITY ADSORBENT FOR PROTEIN UPTAKE

    Institute of Scientific and Technical Information of China (English)

    WANGYongjian; BAIShu; 等

    2001-01-01

    In this article,a spherical chitosan gel crosslinked by epichlorohydrin was prepared.It was then loaded with copper ions to produce a metal chelate affinity adsorbent for protein.The uptake of bovine serum albumin(BSA)by the affinity adsorbent was investigated.and the adsorption capacity for BSA as high as 40mg/g-wet beads was observed.The adsorption equilibrium data was well correlated by the Langmuir equation.The adsorption was considerably affected by pH.In additio.The amount of BSA adsorbed onto the beads decreased with the increasing of aqueous phase ionic strength,so adsorbed BAS can be desorbed by adjusting pH orionic strength of the solution.

  1. An affinity-directed protein missile system for targeted proteolysis

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    Fulcher, Luke J.; Macartney, Thomas; Bozatzi, Polyxeni; Hornberger, Annika; Rojas-Fernandez, Alejandro

    2016-01-01

    The von Hippel–Lindau (VHL) protein serves to recruit the hypoxia-inducible factor alpha (HIF1α) protein under normoxia to the CUL2 E3 ubiquitin ligase for its ubiquitylation and degradation through the proteasome. In this report, we modify VHL to engineer an affinity-directed protein missile (AdPROM) system to direct specific endogenous target proteins for proteolysis in mammalian cells. The proteolytic AdPROM construct harbours a cameloid anti-green fluorescence protein (aGFP) nanobody that is fused to VHL for either constitutive or tetracycline-inducible expression. For target proteins, we exploit CRISPR/Cas9 to rapidly generate human kidney HEK293 and U2OS osteosarcoma homozygous knock-in cells harbouring GFP tags at the VPS34 (vacuolar protein sorting 34) and protein associated with SMAD1 (PAWS1, aka FAM83G) loci, respectively. Using these cells, we demonstrate that the expression of the VHL-aGFP AdPROM system results in near-complete degradation of the endogenous GFP-VPS34 and PAWS1-GFP proteins through the proteasome. Additionally, we show that Tet-inducible destruction of GFP-VPS34 results in the degradation of its associated partner, UVRAG, and reduction in levels of cellular phosphatidylinositol 3-phosphate. PMID:27784791

  2. High-throughput analysis of protein-DNA binding affinity.

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    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  3. APPLICATION OF IMMUNOGLOBULIN-BINDING PROTEINS A, G, L IN THE AFFINITY CHROMATOGRAPHY

    OpenAIRE

    Sviatenko, О.; Gorbatiuk, O.; Vasylchenko, О.

    2014-01-01

    Proteins A, G and L are native or recombinant proteins of microbial origin that bind to mammalian immunoglobulins. Preferably recombinant variants of proteins A, G, L are used in biotechnology for affinity sorbents production. Сomparative characteristics of proteins A, G, L and affinity sorbents on the basis of them, advantages and disadvantages of these proteins application as ligands in the affinity chromatography are done. Analysis of proteins A, G, L properties is presented. Binding speci...

  4. A thermal responsive affinity ligand for precipitation of sialylated proteins

    Directory of Open Access Journals (Sweden)

    Lindsay Arnold

    2016-01-01

    Full Text Available We report here the development of a thermal responsive affinity ligand specific to sialic acid, sialic acid containing oligosaccharides, glycoproteins, and other sialylated glycoconjugates. The ligand is a fusion protein of 40 repeats of pentapeptide of an elastin like polymer (ELP and the 21 kD sialic acid binding domain of a Vibrio cholera neuraminidase (VCNA. For cost-effective synthesis, the fusion protein was targeted to the periplasmic space of an E. coli lpp deletion mutant, resulting in its secretion to the growth medium. A pre-induction heat-shock step at 42 ˚C for 20 minutes was necessary to achieve high level expression of the ligand. Under optimized induction condition (18 ˚C, 0.1 mM IPTG and 48 hours of post-induction cultivation, the ligand was produced to about 100 mg/L. The ligand exhibited a transition temperature of 52 ˚C, which could be depressed to 37 ˚C with the addition of 0.5 M NaCl. Using fetuin as a model sialylated protein, the ligand was applied in an affinity precipitation process to illustrate its potential application in glycoprotein isolation. The ligand captured 100% fetuin from an aqueous solution when the molar ratio of ligand to fetuin was 10 to 1, which was lower than the expected for full titration of sialic acid on the glycoprotein by the lectin. Elution of fetuin from ligand was achieved with PBS buffer containing 2 mM sialic acid. To evaluate how protein and other contaminants influence the recovery of sialylated proteins, CHO medium was spiked into the fetuin solution. The predominant protein species in CHO medium was found to be albumin. Although its removal of over 94% was evident, purified fetuin contained some albumin due to its over-abundance. Additional experiments with albumin contaminant of varying concentrations showed that below 1 mg/L, albumin had no impact on the affinity precipitation, whereas above 10 mg/L, some albumin was co-purified with fetuin. However, even at 50 mg/ml, fetuin

  5. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

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    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  6. Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

    Science.gov (United States)

    O'Brien, Jeffrey; Shea, Kenneth J

    2016-06-21

    Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is

  7. Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

    Science.gov (United States)

    O'Brien, Jeffrey; Shea, Kenneth J

    2016-06-21

    Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is

  8. Interplay between binding affinity and kinetics in protein-protein interactions.

    Science.gov (United States)

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc.

  9. Interplay between binding affinity and kinetics in protein-protein interactions.

    Science.gov (United States)

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  10. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    Science.gov (United States)

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  11. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    Science.gov (United States)

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  12. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    DEFF Research Database (Denmark)

    Nissen, Klaus B; Kedström, Linda Maria Haugaard; Wilbek, Theis S;

    2015-01-01

    related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of...... trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG...... linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic...

  13. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

    Science.gov (United States)

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-01-01

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process. PMID:27438863

  14. Surface Plasmon Resonance for Rapid Screening of Uranyl Affine Proteins

    International Nuclear Information System (INIS)

    A sensitive immunoassay based on SPR analysis was developed to measure uranyl cation (UO22+) affinity for any protein in a free state under physiological conditions. The technique involves immobilization of a specific monoclonal antibody (mAb) raised against UO22+ and 1, 10-phenanthroline-2, 9-dicarboxylic acid (DCP) used as a probe of UO22+ captured by the mAb. Calibration curves were established for accurate determination of UO22+ concentrations with a detection limit of 7 nM. The remaining free UO22+ could be accurately quantified from the different protein-metal equilibrium and a dose-response curve established for KD determination. This generic method was applied not only to proteins such as transferrin and albumin but also to small phosphonated ligands. Its robustness allows the fast UO22+ KD determination of any kind of macromolecules and small ligands using very few amount of compounds, thus opening new prospects in the field of uranium toxicity. (authors)

  15. Predicting the Impact of Missense Mutations on Protein-Protein Binding Affinity.

    Science.gov (United States)

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-04-01

    The crucial prerequisite for proper biological function is the protein's ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein-protein binding is critical for a wide range of biomedical applications. Here, we report an efficient computational approach for predicting the effect of single and multiple missense mutations on protein-protein binding affinity. It is based on a well-tested simulation protocol for structure minimization, modified MM-PBSA and statistical scoring energy functions with parameters optimized on experimental sets of several thousands of mutations. Our simulation protocol yields very good agreement between predicted and experimental values with Pearson correlation coefficients of 0.69 and 0.63 and root-mean-square errors of 1.20 and 1.90 kcal mol(-1) for single and multiple mutations, respectively. Compared with other available methods, our approach achieves high speed and prediction accuracy and can be applied to large datasets generated by modern genomics initiatives. In addition, we report a crucial role of water model and the polar solvation energy in estimating the changes in binding affinity. Our analysis also reveals that prediction accuracy and effect of mutations on binding strongly depends on the type of mutation and its location in a protein complex. PMID:24803870

  16. A community standard format for the representation of protein affinity reagents

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Orchard, Sandra; Bertinetti, Daniela;

    2010-01-01

    Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often...

  17. Dual-tagging system for the affinity purification of mammalian protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL

    2007-01-01

    Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

  18. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    OpenAIRE

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  19. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua;

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  20. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  1. Statistically Inferring Protein-Protein Asociations with Affinity Isolation LC-MS/MS Assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L.; Anderson, Kevin K.; Hurst, G. B.; Daly, Don S.; Pelletier, Dale A.; Cannon, William R.; Auberry, Deanna L.; Schmoyer, Denise D.; McDonald, W. Hayes; White, Amanda M.; Hooker, Brian S.; Victry, Kristin D.; Buchanan, M. V.; Kery, Vladimir; Wiley, H. S.

    2007-09-30

    Affinity isolation of protein complexes followed by protein identification by LC-MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein-protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes’ Odds estimation. We then applied our LRT-Bayes’ algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. The algorithm can discriminate against a background of prey proteins that are detected in association with a large number of baits as an artifact of the measurement. We conclude that the experimental protocol including the LRT-Bayes’ algorithm produces results with high confidence but moderate sensitivity. We also found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.

  2. Computational protein design suggests that human PCNA-partner interactions are not optimized for affinity.

    Science.gov (United States)

    Fridman, Yearit; Gur, Eyal; Fleishman, Sarel J; Aharoni, Amir

    2013-02-01

    Increasing the affinity of binding proteins is invaluable for basic and applied biological research. Currently, directed protein evolution experiments are the main approach for generating such proteins through the construction and screening of large mutant libraries. Proliferating cell nuclear antigen (PCNA) is an essential hub protein that interacts with many different partners to tightly regulate DNA replication and repair in all eukaryotes. Here, we used computational design to generate human PCNA mutants with enhanced affinity for several different partners. We identified double mutations in PCNA, outside the main partner binding site, that were predicted to increase PCNA-partner binding affinities compared to the wild-type protein by forming additional hydrophobic interactions with conserved residues in the PCNA partners. Affinity increases were experimentally validated with four different PCNA partners, demonstrating that computational design can reveal unexpected regions where affinity enhancements in natural systems are possible. The designed PCNA mutants can be used as a valuable tool for further examination of the regulation of PCNA-partner interactions during DNA replication and repair both in vitro and in vivo. More broadly, the ability to engineer affinity increases toward several PCNA partners suggests that interaction affinity is not an evolutionarily optimized trait of this system. PMID:23011891

  3. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    Science.gov (United States)

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  4. Statistically Inferring Protein-Protein Assocations with Affinity isolation LC-MS/MS assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L. [Montana State University; Anderson, Kevin K. [Pacific Northwest National Laboratory (PNNL); Hurst, Gregory {Greg} B [ORNL; Daly, Don S. [Pacific Northwest National Laboratory (PNNL); Pelletier, Dale A [ORNL; Cannon, Bill [Pacific Northwest National Laboratory (PNNL); Auberry, Deanna L [ORNL; Schmoyer, Denise D [ORNL; McDonald, W Hayes [ORNL; White, Amanda M. [Pacific Northwest National Laboratory (PNNL); Hooker, Brian [Pacific Northwest National Laboratory (PNNL); Victry, Kristin D [Pacific Northwest National Laboratory (PNNL); Buchanan, Michelle V [ORNL; Kerry, Vladimir [Pacific Northwest National Laboratory (PNNL); Wiley, Steven [Pacific Northwest National Laboratory (PNNL); Doktycz, Mitchel John [ORNL

    2007-01-01

    Affinity isolation of protein complexes followed by protein identification by LC-MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein-protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes' Odds estimation. We then applied our LRT-Bayes' algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. We conclude that the experimental protocol including the LRT-Bayes' algorithm produces results with high confidence but moderate sensitivity. We also found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.

  5. Statistically Inferring Protein-Protein Associations with Affinity Isolation LC-MS/MS Assays

    Energy Technology Data Exchange (ETDEWEB)

    Sharp, Julia L. [Montana State University; Anderson, Kevin K. [Pacific Northwest National Laboratory (PNNL); Daly, Don S. [Pacific Northwest National Laboratory (PNNL); Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Cannon, Bill [Pacific Northwest National Laboratory (PNNL); Auberry, Deanna L [ORNL; Schmoyer, Denise D [ORNL; McDonald, W Hayes [ORNL; White, Amanda M. [Pacific Northwest National Laboratory (PNNL); Hooker, Brian [Pacific Northwest National Laboratory (PNNL); Victry, Kristin D [Pacific Northwest National Laboratory (PNNL); Buchanan, Michelle V [ORNL; Kerry, Vladimir [Pacific Northwest National Laboratory (PNNL); Wiley, Steven [Pacific Northwest National Laboratory (PNNL)

    2007-01-01

    Affinity isolation of protein complexes followed by protein identification by LC-MS/MS is an increasingly popular approach for mapping protein interactions. However, systematic and random assay errors from multiple sources must be considered to confidently infer authentic protein-protein interactions. To address this issue, we developed a general, robust statistical method for inferring authentic interactions from protein prey-by-bait frequency tables using a binomial-based likelihood ratio test (LRT) coupled with Bayes Odds estimation. We then applied our LRT-Bayes algorithm experimentally using data from protein complexes isolated from Rhodopseudomonas palustris. Our algorithm, in conjunction with the experimental protocol, inferred with high confidence authentic interacting proteins from abundant, stable complexes, but few or no authentic interactions for lower-abundance complexes. We conclude that the experimental protocol including the LRT-Bayes algorithm produces results with high confidence but moderate sensitivity. We also found that Monte Carlo simulation is a feasible tool for checking modeling assumptions, estimating parameters, and evaluating the significance of results in protein association studies.

  6. Protein-ligand binding affinities from large-scale quantum mechanical simulations

    OpenAIRE

    Fox, Stephen J.

    2012-01-01

    The accurate prediction of protein-drug binding affinities is a major aim of computational drug optimisation and development. A quantitative measure of binding affinity is provided by the free energy of binding, and such calculations typically require extensive configurational sampling of entities such as proteins with thousands of atoms. Current binding free energy methods use force fields to perform the configurational sampling and to compute interaction energies. Due to the empirical natur...

  7. Affinity Pull-Down of Proteins Using Anti-FLAG M2 Agarose Beads

    Science.gov (United States)

    Gerace, Erica; Moazed, Danesh

    2016-01-01

    FLAG is an affinity tag widely used for rapid and highly specific one-step protein purification. Native elution of protein from anti-FLAG antibody resins allows the identification of protein and nucleic acid binding partners and functional analysis using biochemical activity assays. PMID:26096505

  8. Tandem affinity purification to identify cytosolic and nuclear gβγ-interacting proteins.

    Science.gov (United States)

    Campden, Rhiannon; Pétrin, Darlaine; Robitaille, Mélanie; Audet, Nicolas; Gora, Sarah; Angers, Stéphane; Hébert, Terence E

    2015-01-01

    It has become clear in recent years that the Gβγ subunits of heterotrimeric proteins serve broad roles in the regulation of cellular activity and interact with many proteins in different subcellular locations including the nucleus. Protein affinity purification is a common method to identify and confirm protein interactions. When used in conjugation with mass spectrometry it can be used to identify novel protein interactions with a given bait protein. The tandem affinity purification (TAP) technique identifies partner proteins bound to tagged protein bait. Combined with protocols to enrich the nuclear fraction of whole cell lysate through sucrose cushions, TAP allows for purification of interacting proteins found specifically in the nucleus. Here we describe the use of the TAP technique on cytosolic and nuclear lysates to identify candidate proteins, through mass spectrometry, that bind to Gβ1 subunits.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; Liu, Yie [ORNL; Wang, Yisong [ORNL

    2009-01-01

    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 utilizes 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 have demonstrated 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.

  11. Semi-continuous protein fractionating using Affinity Cross-Flow Filtration

    NARCIS (Netherlands)

    Borneman, Z.; Zhang, W.; Boomgaard, van den Th.; Smolders, C.A.

    2002-01-01

    Protein purification by means of downstream processing is increasingly important. At the University of Twente a semi-continuous process is developed for the isolation of BSA out of crude protein mixtures. For this purpose an automated Affinity Cross-Flow Filtration, ACFF, process is developed. This

  12. In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

    Science.gov (United States)

    Diamante, Letizia; Gatti-Lafranconi, Pietro; Schaerli, Yolanda; Hollfelder, Florian

    2013-10-01

    The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity. The utility of the BeSD in directed evolution is validated by library selections of randomized peptide sequences for binding to the anti-hemagglutinin (HA) antibody that proceed with enrichments in excess of 10(3) and lead to the isolation of high-affinity HA-tags within one round of flow cytometric screening. On-bead K(d) measurements suggest that the selected tags have affinities in the low nanomolar range. In contrast to other display systems (such as ribosome, mRNA and phage display) that are limited to affinity panning selections, BeSD possesses the ability to screen and rank binders by their affinity in vitro, a feature that hitherto has been exclusive to in vivo multivalent cell display systems (such as yeast display).

  13. Fluorous-assisted metal chelate affinity extraction technique for analysis of protein kinase activity.

    Science.gov (United States)

    Hayama, Tadashi; Kiyokawa, Ena; Yoshida, Hideyuki; Imakyure, Osamu; Yamaguchi, Masatoshi; Nohta, Hitoshi

    2016-08-15

    We have developed a fluorous affinity-based extraction method for measurement of protein kinase activity. In this method, a fluorescent peptide substrate was phosphorylated by a protein kinase, and the obtained phosphopeptide was selectively captured with Fe(III)-immobilized perfluoroalkyliminodiacetic acid reagent via a metal chelate affinity technique. Next, the captured phosphopeptide was selectively extracted into a fluorous solvent mixture, tetradecafluorohexane and 1H,1H,2H,2H-tridecafluoro-1-n-octanol (3:1, v/v), using the specificity of fluorous affinity (fluorophilicity). In contrast, the remained substrate peptide in the aqueous (non-fluorous) phase was easily measured fluorimetrically. Finally, the enzyme activity could be assayed by measuring the decrease in fluorescence. The feasibility of this method was demonstrated by applying the method for measurement of the activity of cAMP-dependent protein kinase (PKA) using its substrate peptide (kemptide) pre-labeled with carboxytetramethylrhodamine (TAMRA).

  14. Presence of proteolipid protein in coelacanth brain myelin demonstrates tetrapod affinities and questions a chondrichthyan association.

    Science.gov (United States)

    Waehneldt, T V; Malotka, J

    1989-06-01

    The protein and glycoprotein compositions of CNS myelin from the living coelacanth (Latimeria chalumnae) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An unglycosylated component of 25 kilodaltons showed substantially stronger immunoblot reactivity with antibodies against mammalian proteolipid protein (PLP) than lungfish glycosylated PLP. DM-20 (intermediate protein) was not detectable in either fish. The presence of unglycosylated PLP in CNS myelin of the actinistian coelacanth contradicts an association with cartilaginous fishes but supports tetrapod affinities closer than those of lungfish.

  15. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  16. Parameterization of an effective potential for protein-ligand binding from host-guest affinity data.

    Science.gov (United States)

    Wickstrom, Lauren; Deng, Nanjie; He, Peng; Mentes, Ahmet; Nguyen, Crystal; Gilson, Michael K; Kurtzman, Tom; Gallicchio, Emilio; Levy, Ronald M

    2016-01-01

    Force field accuracy is still one of the "stalemates" in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein-ligand binding, organic host-guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host-guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein-ligand binding in two drug targets against the HIV-1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics-based binding free energy models can be used to evaluate and optimize force fields for protein-ligand systems. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26256816

  17. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    Science.gov (United States)

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions. PMID:17223789

  18. Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

    Directory of Open Access Journals (Sweden)

    Miriam S. Goyder

    2012-04-01

    Full Text Available We present a top down separation platform for yeast ribosomalproteins using affinity chromatography and capillary electrophoresiswhich is designed to allow deposition of proteins ontoa substrate. FLAG tagged ribosomes were affinity purified, andrRNA acid precipitation was performed on the ribosomes followedby capillary electrophoresis to separate the ribosomalproteins. Over 26 peaks were detected with excellent reproducibility(<0.5% RSD migration time. This is the first reportedseparation of eukaryotic ribosomal proteins using capillaryelectrophoresis. The two stages in this workflow, affinity chromatographyand capillary electrophoresis, share the advantagesthat they are fast, flexible and have small sample requirementsin comparison to more commonly used techniques. This methodis a remarkably quick route from cell to separation that hasthe potential to be coupled to high throughput readout platformsfor studies of the ribosomal proteome. [BMB reports2012; 45(4: 233-238

  19. Photoaffinity labelling of high affinity dopamine binding proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  20. Low levels of high-affinity growth hormone-binding protein in African pygmies.

    Science.gov (United States)

    Baumann, G; Shaw, M A; Merimee, T J

    1989-06-29

    The cause of growth hormone resistance and short stature in African Pygmies in unknown. Low levels of insulin-like growth factor 1 that fail to respond to growth hormone suggest a possible deficiency of growth hormone receptors. The high-affinity growth hormone-binding protein is a fragment of the growth hormone receptor and may be an indicator of the number of receptors in tissues. We measured growth hormone-binding activity in plasma from 20 pygmies and 12 control subjects (7 white Americans and 5 non-Pygmy black Africans of normal stature). Growth hormone binding to the high-affinity binding protein was significantly reduced in the Pygmies as compared with the controls (mean +/- SD, 6.50 +/- 2.33 percent vs. 12.95 +/- 3.95 percent bound per 160 microliters of plasma; P less than 0.001); however, there was substantial overlap between the values for Pygmies and controls. Growth hormone binding to the low-affinity binding protein was similar in Pygmies and control subjects. We conclude that Pygmies have low levels of high-affinity growth hormone-binding protein in their plasma, which may indicate a reduced number of growth hormone receptors in their tissues. The finding may help explain the resistance to growth in Pygmies, but there may be additional reasons, related to the receptors or not, for their short stature.

  1. Predicting the Impact of Missense Mutations on Protein–Protein Binding Affinity

    OpenAIRE

    Li, Minghui; Petukh, Marharyta; Alexov, Emil; Panchenko, Anna R

    2014-01-01

    The crucial prerequisite for proper biological function is the protein’s ability to establish highly selective interactions with macromolecular partners. A missense mutation that alters the protein binding affinity may cause significant perturbations or complete abolishment of the function, potentially leading to diseases. The availability of computational methods to evaluate the impact of mutations on protein–protein binding is critical for a wide range of biomedical applications. Here, we r...

  2. A dual-tagging system for the affinity purification of mammalian protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Huang, Ying [ORNL; Wu, Jun [ORNL; Liu, Yie [National Institute on Aging, Baltimore; Wang, Yisong [ORNL

    2007-01-01

    One popular method to elucidate protein-protein interactions involves the native co-purification of an affinity tagged protein and its interacting partners, which are subsequently identified through mass spectrometry (MS) (1). Although straightforward, reproducible, and broadly employed, this strategy is hampered by the efficacy of protein recoveries both in terms of sensitivity and specificity. This is especially pertinent to methodologies that employ a single-step of purification, where suboptimal enrichment of the bait protein and its partners over background can lead to masking of their signals. Although improvements to MS instrumentation generally increase peptide detection sensitivities, the problem of specificity, i.e. distinguishing specific from non-specific interacting proteins, remains. Thus ultimately, the limiting factor in the identification of specific interacting proteins lies with the purification itself. An effort to resolve this specificity issue has been made with the introduction of the Tandem Affinity Purification (TAP) tag. This construct consists of an IgG-binding domain and calmodulin binding peptide domain separated by a tobacco etch virus (TEV) protease cleavage site (2). The TAP method was originally developed in yeast and has best demonstrated its utility in the systematic identification of numerous multiprotein complexes in the yeast proteome (3). Although modifications to the original TAP have been successful in examining the protein networks of mammalian cells (4-7), the strategy offers a relatively low yield of bait and specific interacting proteins (8), and the success rate are usually on case-by-case basis. In addition, problems inherent to any protein tagging strategy remain, such as variable exposure of the affinity tag, disruption of the bait protein's ability to fold properly, steric exclusion of interacting partners, and/or ectopic overexpression of the fusion protein, which can lead to complications in both the

  3. Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

    Science.gov (United States)

    Millward, Steven W; Fiacco, Stephen; Austin, Ryan J; Roberts, Richard W

    2007-09-21

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.

  4. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions.

    Science.gov (United States)

    Altschuler, Sarah E; Lewis, Karen A; Wuttke, Deborah S

    2013-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

  5. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    Directory of Open Access Journals (Sweden)

    Sarah E. Altschuler

    2013-09-01

    Full Text Available The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

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

    Science.gov (United States)

    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. PMID:27436096

  7. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong

    2011-10-01

    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

  8. Evaluation of Affinity-Tagged Protein Expression Strategies using Local and Global Isotope Ratio Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Hervey, IV, William Judson [ORNL; Khalsa-Moyers, Gurusahai K [ORNL; Lankford, Patricia K [ORNL; Owens, Elizabeth T [ORNL; McKeown, Catherine K [ORNL; Lu, Tse-Yuan S [ORNL; Foote, Linda J [ORNL; Morrell-Falvey, Jennifer L [ORNL; McDonald, W Hayes [ORNL; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL

    2009-01-01

    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 by 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

  9. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  10. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

    Science.gov (United States)

    Peng, Tao; Free, Paul; Fernig, David G.; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of

  11. PBSA_E: A PBSA-Based Free Energy Estimator for Protein-Ligand Binding Affinity.

    Science.gov (United States)

    Liu, Xiao; Liu, Jinfeng; Zhu, Tong; Zhang, Lujia; He, Xiao; Zhang, John Z H

    2016-05-23

    Improving the accuracy of scoring functions for estimating protein-ligand binding affinity is of significant interest as well as practical utility in drug discovery. In this work, PBSA_E, a new free energy estimator based on the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) descriptors, has been developed. This free energy estimator was optimized using high-quality experimental data from a training set consisting of 145 protein-ligand complexes. The method was validated on two separate test sets containing 121 and 130 complexes. Comparison of the binding affinities predicted using the present method with those obtained using three popular scoring functions, i.e., GlideXP, GlideSP, and SYBYL_F, demonstrated that the PBSA_E method is more accurate. This new energy estimator requires a MM/PBSA calculation of the protein-ligand binding energy for a single complex configuration, which is typically obtained by optimizing the crystal structure. The present study shows that PBSA_E has the potential to become a robust tool for more reliable estimation of protein-ligand binding affinity in structure-based drug design. PMID:27088302

  12. Relating the shape of protein binding sites to binding affinity profiles: is there an association?

    Directory of Open Access Journals (Sweden)

    Bitter István

    2010-10-01

    Full Text Available Abstract Background Various pattern-based methods exist that use in vitro or in silico affinity profiles for classification and functional examination of proteins. Nevertheless, the connection between the protein affinity profiles and the structural characteristics of the binding sites is still unclear. Our aim was to investigate the association between virtual drug screening results (calculated binding free energy values and the geometry of protein binding sites. Molecular Affinity Fingerprints (MAFs were determined for 154 proteins based on their molecular docking energy results for 1,255 FDA-approved drugs. Protein binding site geometries were characterized by 420 PocketPicker descriptors. The basic underlying component structure of MAFs and binding site geometries, respectively, were examined by principal component analysis; association between principal components extracted from these two sets of variables was then investigated by canonical correlation and redundancy analyses. Results PCA analysis of the MAF variables provided 30 factors which explained 71.4% of the total variance of the energy values while 13 factors were obtained from the PocketPicker descriptors which cumulatively explained 94.1% of the total variance. Canonical correlation analysis resulted in 3 statistically significant canonical factor pairs with correlation values of 0.87, 0.84 and 0.77, respectively. Redundancy analysis indicated that PocketPicker descriptor factors explain 6.9% of the variance of the MAF factor set while MAF factors explain 15.9% of the total variance of PocketPicker descriptor factors. Based on the salient structures of the factor pairs, we identified a clear-cut association between the shape and bulkiness of the drug molecules and the protein binding site descriptors. Conclusions This is the first study to investigate complex multivariate associations between affinity profiles and the geometric properties of protein binding sites. We found that

  13. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    Science.gov (United States)

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  14. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  15. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    Science.gov (United States)

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  16. Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities

    Science.gov (United States)

    Yao, Yuyu; Richards, Michele R.; Kitova, Elena N.; Klassen, John S.

    2016-03-01

    The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)- d-GlcNAc, a single chain variable fragment and α- d-Gal-(1→2)-[α- d-Abe-(1→3)]-α- d-Man-OCH3, cholera toxin B subunit homopentamer with β- d-Gal-(1→3)-β- d-GalNAc-(1→4)[α- d-Neu5Ac-(2→3)]-β- d-Gal-(1→4)-β- d-Glc, and a fragment of galectin 3 and α- l-Fuc-(1→2)-β- d-Gal-(1→3)-β- d-GlcNAc-(1→3)-β- d-Gal-(1→4)-β- d-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

  17. NiCoMnO4: A Bifunctional Affinity Probe for His-Tagged Protein Purification and Phosphorylation Sites Recognition.

    Science.gov (United States)

    Qi, Xiaoyue; Chen, Long; Zhang, Chaoqun; Xu, Xinyuan; Zhang, Yiding; Bai, Yu; Liu, Huwei

    2016-07-27

    A bifunctional affinity probe NiCoMnO4 was designed and prepared with controllable morphology and size using facile methods. It was observed that the probe could be applied in His-tagged proteins purification and phosphopeptides enrichment simply through the buffer modulation. NiCoMnO4 particles showed satisfactory cycling performance for His-tagged proteins purification and broad pH-tolerance of loading buffer for phosphopeptides affinity. Therefore, a high-throughput, cost-effective, and efficient protein/peptide purification method was developed within 10 min based on the novel bifunctional affinity probe. PMID:27381638

  18. Tetrapyrrole binding affinity of the murine and human p22HBP heme-binding proteins.

    Science.gov (United States)

    Micaelo, Nuno M; Macedo, Anjos L; Goodfellow, Brian J; Félix, Vítor

    2010-11-01

    We present the first systematic molecular modeling study of the binding properties of murine (mHBP) and human (hHBP) p22HBP protein (heme-binding protein) with four tetrapyrrole ring systems belonging to the heme biosynthetic pathway: iron protoporphyrin IX (HEMIN), protoporphyrin IX (PPIX), coproporphyrin III (CPIII), coproporphyrin I (CPI). The relative binding affinities predicted by our computational study were found to be similar to those observed experimentally, providing a first rational structural analysis of the molecular recognition mechanism, by p22HBP, toward a number of different tetrapyrrole ligands. To probe the structure of these p22HBP protein complexes, docking, molecular dynamics and MM-PBSA methodologies supported by experimental NMR ring current shift data have been employed. The tetrapyrroles studied were found to bind murine p22HBP with the following binding affinity order: HEMIN> PPIX> CPIII> CPI, which ranged from -22.2 to -6.1 kcal/mol. In general, the protein-tetrapyrrole complexes are stabilized by non-bonded interactions between the tetrapyrrole propionate groups and basic residues of the protein, and by the preferential solvation of the complex compared to the unbound components. PMID:20800521

  19. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2016-03-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  20. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  1. Isolation and identification of actin-binding proteins in Plasmodium falciparum by affinity chromatography

    Directory of Open Access Journals (Sweden)

    Claudia Forero

    2000-06-01

    Full Text Available The invasion of the erythrocyte by Plasmodium falciparum depends on the ability of the merozoite to move through the membrane invagination. This ability is probably mediated by actin dependent motors. Using affinity columns with G-actin and F-actin we isolated actin binding proteins from the parasite. By immunoblotting and immunoprecipitation with specific antibodies we identified the presence of tropomyosin, myosin, a-actinin, and two different actins in the eluate corresponding to F-actin binding proteins. In addition to these, a 240-260 kDa doublet, different in size from the erythrocyte spectrin, reacted with an antibody against human spectrin. All the above mentioned proteins were metabolically radiolabeled when the parasite was cultured with 35S-methionine. The presence of these proteins in P. falciparum is indicative of a complex cytoskeleton and supports the proposed role for an actin-myosin motor during invasion.

  2. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    International Nuclear Information System (INIS)

    CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance

  3. Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

    Science.gov (United States)

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

    Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

  4. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  5. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    OpenAIRE

    Kaake, Robyn M; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand ho...

  6. Metal binding affinity and structural properties of calmodulin-like protein 14 from Arabidopsis thaliana.

    Science.gov (United States)

    Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Giorgetti, Alejandro; Dominici, Paola; Astegno, Alessandra

    2016-08-01

    In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd ∼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by ∼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role. PMID:27124620

  7. Structure and Energetic Contributions of a Designed Modular Peptide-Binding Protein with Picomolar Affinity.

    Science.gov (United States)

    Hansen, Simon; Tremmel, Dirk; Madhurantakam, Chaithanya; Reichen, Christian; Mittl, Peer R E; Plückthun, Andreas

    2016-03-16

    Natural armadillo repeat proteins (nArmRP) like importin-α or β-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 Å resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats. PMID:26878586

  8. Specificity and affinity of phenosafranine protein adduct: Insights from biophysical aspects

    Energy Technology Data Exchange (ETDEWEB)

    Ding Fei [Department of Chemistry, China Agricultural University, Beijing 100193 (China); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (United States); Zhang Li; Sun Ye [Department of Chemistry, China Agricultural University, Beijing 100193 (China); Diao Jianxiong [College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193 (China); Yang Xinling [Department of Chemistry, China Agricultural University, Beijing 100193 (China); Sun Ying, E-mail: sunying@cau.edu.cn [College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193 (China); Zhang Li, E-mail: zhli.work@gmail.com [Key Laboratory of Pesticide Chemistry and Application Technology, Ministry of Agriculture, Department of Applied Chemistry, China Agricultural University, Beijing 100193 (China)

    2012-03-15

    Phenosafranine is a toxic and recalcitrant compound, whose capacity to intercalate with double stranded DNA has been shown. In this contribution, a biophysical discuss on the conjugation of phenosafranine with two model proteins human serum albumin (HSA) and lysozyme (Lys) has been identified utilizing a combination of molecular modeling, steady state and time-resolved fluorescence and circular dichroism (CD) approaches. The accurate binding domain of phenosafranine in protein has been characterized from molecular modeling, subdomain IIIA of HSA and Trp-62, Trp-63 and Trp-108 residues of Lys was designated to possess high-affinity for this compound, the dominant forces in the protein-phenosafranine adduct are hydrogen bonds and {pi}-{pi} interactions, but hydrophobic interactions between dye and Lys are also not exclude. The data of fluorescence displayed that the complex of phenosafranine with protein produces quenching through static property, this corroborates the time-resolved fluorescence results that the ground state complex formation with a moderate affinity of 10{sup 4} M{sup -1}. Moreover, via synchronous fluorescence, CD and three-dimensional fluorescence we indicated some extent of polypeptide chain of protein partially unfolding upon conjugation with phenosafranine. Through this work, we anticipate it can supply salient clues on the toxicological action of phenosafranine and other azines, which have analogous configuration with phenosafranine. - Highlights: Black-Right-Pointing-Pointer Our study supply salient clues on the toxicological action of toxic phenosafranine. Black-Right-Pointing-Pointer Phenosafranine is situated within subdomain IIIA, Sudlow's site II on HSA. Black-Right-Pointing-Pointer Trp-62, Trp-63 and Trp-108 residues on the Lys molecule are all close to dye. Black-Right-Pointing-Pointer Static property of the phenosafranine induced quenching of protein Trp fluorescence. Black-Right-Pointing-Pointer Protein spatial structure proved

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

    Directory of Open Access Journals (Sweden)

    Hua-zhen Wang

    Full Text Available 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.

  10. Phycobiliprotein fusion proteins: versatile intensely fluorescent constructs

    Science.gov (United States)

    Glazer, Alexander N.; Cai, Yuping A.; Tooley, Aaron J.

    2004-06-01

    Since 1982, phycobiliproteins have served as fluorescent labels in a wide variety of cell and molecule analyses. The exceptional spectroscopic properties of these labels include very high absorbance coefficients and quantum yields, and large Stokes shifts. The spectroscopic diversity of these reagents is restricted to a subset of naturally occurring phycobiliproteins with stable assembly states in vitro, whose target specificity is generated by chemical conjugation to proteins or small molecules. The latter step generates heterogeneity. These limitations have been overcome by expressing various recombinant phycobiliprotein constructs in the cyanobacterium Anabaena sp. PCC7120. Modular recombinant phycobiliprotein-based labels were constructed with some or all of the following features (a) an affinity purification tag; (b) a stable oligomerization domain (to maintain stable higher order assemblies of the phycobiliprotein monomers at very low protein concentration); (c) a biospecific recognition domain. Such phycobiliprotein constructs are readily purified from crude cell extracts by affinity chromatography and used directly as fluorescent labels. To generate constructs for intracellular in vivo labeling, the entire pathways for the biosynthesis of the His-tagged holo- α (phycocyanobilin-bearing) subunit of phycocyanin (emission max. 641 nm) and of the His-tagged holo-α (phycobiliviolin-bearing) subunit of phycoerythrocyanin (emission max. 582 nm) were reconstituted in Escherichia coli.

  11. High Affinity Immobilization of Proteins Using the CrAsH/TC Tag

    Directory of Open Access Journals (Sweden)

    Janine Schulte-Zweckel

    2016-06-01

    Full Text Available Protein microarrays represent important tools for biomedical analysis. We have recently described the use of the biarsenical-tetracysteine (TC tag for the preparation of protein microarrays. The unique feature of this tag enables the site-specific immobilization of TC-containing proteins on biarsenical-modified surfaces, resulting in a fluorescence enhancement that allows the direct quantification of the immobilized proteins. Moreover, the reversibility of the binding upon incubation with large quantities of thiols permits the detachment of the proteins from the surface, thereby enabling recovery of the substrate to extend the life time of the slide. Herein, we describe our recent results that further extend the applicability of the CrAsH/TC tag to the fabrication of biochips. With this aim, the immobilization of proteins on surfaces has been investigated using two different spacers and two TC tags, the minimal TC sequence (CCPGCC and an optimized motif (FLNCCPGCCMEP. While the minimal peptide motif enables a rapid recycling of the slide, the optimized TC sequence reveals an increased affinity due to its greater resistance to displacement by thiols. Moreover, the developed methodology was applied to the immobilization of proteins via on-chip ligation of recombinant protein thioesters.

  12. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    Science.gov (United States)

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  13. Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

    Energy Technology Data Exchange (ETDEWEB)

    Der, Bryan S.; Machius, Mischa; Miley, Michael J.; Mills, Jeffrey L.; Szyperski, Thomas; Kuhlman, Brian (UNC); (Buffalo)

    2015-10-15

    Computationally designing protein-protein interactions with high affinity and desired orientation is a challenging task. Incorporating metal-binding sites at the target interface may be one approach for increasing affinity and specifying the binding mode, thereby improving robustness of designed interactions for use as tools in basic research as well as in applications from biotechnology to medicine. Here we describe a Rosetta-based approach for the rational design of a protein monomer to form a zinc-mediated, symmetric homodimer. Our metal interface design, named MID1 (NESG target ID OR37), forms a tight dimer in the presence of zinc (MID1-zinc) with a dissociation constant <30 nM. Without zinc the dissociation constant is 4 {micro}M. The crystal structure of MID1-zinc shows good overall agreement with the computational model, but only three out of four designed histidines coordinate zinc. However, a histidine-to-glutamate point mutation resulted in four-coordination of zinc, and the resulting metal binding site and dimer orientation closely matches the computational model (C{alpha} rmsd = 1.4 {angstrom}).

  14. Identification of protein partners in mycobacteria using a single-step affinity purification method.

    Directory of Open Access Journals (Sweden)

    Przemysław Płociński

    Full Text Available Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis, is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP, protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH, making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners.

  15. Reaction of Native and Denatured Brucella abortus (S19 Proteins with Antibody Using Affinity Chromatography and Immunoblotting

    Directory of Open Access Journals (Sweden)

    R. Karimi

    2005-01-01

    Full Text Available Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure.In this study proteins of Brucella abortus (S19 was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG fraction of the sera was precipitated by half saturation of ammonium sulfate and linked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE. Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting.Upon the results of affinity chromatography and immunoblotting, Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westernblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.

  16. Structures of the Ultra-High-Affinity Protein-Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa.

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A; Lowe, Edward D; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W; Cogdell, Richard J; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-08-28

    How ultra-high-affinity protein-protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase-Im interaction is a model system for the study of high-affinity protein-protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase-Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase-ImS2 and pyocin AP41 DNase-ImAP41. These structures represent divergent DNase-Im subfamilies and are important in extending our understanding of protein-protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase-Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase-Immunity pairs that appear to underpin the split of this family into two distinct groups.

  17. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    Directory of Open Access Journals (Sweden)

    Wenping Gong

    Full Text Available Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs, which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  18. Affinity Purification of O-Acetylserine(thiollyase from Chlorella sorokiniana by Recombinant Proteins from Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Giovanna Salbitani

    2014-08-01

    Full Text Available In the unicellular green alga Chlorella sorokiniana (211/8 k, the protein O-acetylserine(thiollyase (OASTL, representing the key-enzyme in the biosynthetic cysteine pathway, was isolated and purified to apparent homogeneity. The purification was carried out in cells grown in the presence of all nutrients or in sulphate (S deprived cells. After 24 h of S-starvation, a 17-fold increase in the specific activity of OASTL was measured. In order to enable the identification of OASTL proteins from non-model organisms such as C. sorokiniana, the recombinant his-tagged SAT5 protein from Arabidopsis thaliana was immobilized by metal chelate chromatography. OASTL proteins from C. sorokiniana were affinity purified in one step and activities were enhanced 29- and 41-fold, from S-sufficient and S-starved (24 h cells, respectively. The successful application of SAT/OASTL interaction for purification confirms for the first time the existence of the cysteine synthase complexes in microalgae. The purified proteins have apparent molecular masses between 32–34 kDa and are thus slightly larger compared to those found in Arabidopsis thaliana and other vascular plants. The enhanced OASTL activity in S-starved cells can be attributed to increased amounts of plastidic and the emergence of cytosolic OASTL isoforms. The results provide proof-of-concept for the biochemical analysis of the cysteine synthase complex in diverse microalgal species.

  19. Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Petersen, Steen Vang; Jacobsen, Christian;

    2006-01-01

    Haptoglobin-related protein (Hpr) is a primate-specific plasma protein associated with apolipoprotein L-I (apoL-I)-containing high-density lipoprotein (HDL) particles shown to be a part of the innate immune defense. Despite the assumption hitherto that Hpr does not bind to hemoglobin, the present...

  20. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Byung-Kwon [University of Tennessee, Knoxville (UTK); Jung, Kyung-Sik [University of Tennessee, Knoxville (UTK); Son, Cagdas D [ORNL; Kim, Heejung [University of Tennessee, Knoxville (UTK); Verberkmoes, Nathan C [ORNL; Arshava, Boris [College of Staten Island; Naider, Fred [College of Staten Island; Becker, Jeffrey Marvin [ORNL

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  1. Development of an automated mid-scale parallel protein purification system for antibody purification and affinity chromatography.

    Science.gov (United States)

    Zhang, Chi; Long, Alexander M; Swalm, Brooke; Charest, Ken; Wang, Yan; Hu, Jiali; Schulz, Craig; Goetzinger, Wolfgang; Hall, Brian E

    2016-12-01

    Protein purification is often a bottleneck during protein generation for large molecule drug discovery. Therapeutic antibody campaigns typically require the purification of hundreds of monoclonal antibodies (mAbs) during the hybridoma process and lead optimization. With the increase in high-throughput cloning, faster DNA sequencing, and the use of parallel protein expression systems, a need for high-throughput purification approaches has evolved, particularly in the midsize range between 20 ml and 100 ml. To address this we modified a four channel Gilson solid phase extraction system (referred to as MG-SPE) with switching valves and sample holding loops to be able to perform standard affinity purification using commercially available columns and micro-titer format deep well blocks. By running 4 samples in parallel, the MG-SPE has the capacity to purify up to 24 samples of greater than 50 ml each using a single-step affinity purification protocol or a two-step protocol consisting of affinity chromatography followed by desalting/buffer exchange overnight (∼12 h run time). Our evaluation of affinity purification using mAbs and Fc-fusion proteins from mammalian cell supernatants demonstrates that the MG-SPE compared favorably with industry standard systems for both protein quality and yield. Overall the system is simple to operate and fills a void in purification processes where a simple, efficient, automated system is needed for affinity purification of midsize research samples.

  2. A protein engineered to bind uranyl selectively and with femtomolar affinity

    Science.gov (United States)

    Zhou, Lu; Bosscher, Mike; Zhang, Changsheng; Özçubukçu, Salih; Zhang, Liang; Zhang, Wen; Li, Charles J.; Liu, Jianzhao; Jensen, Mark P.; Lai, Luhua; He, Chuan

    2014-03-01

    Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM) however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

  3. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    Energy Technology Data Exchange (ETDEWEB)

    Raza, H.; Chung, W.L.; Mukhtar, H. (Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, OH (USA))

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  4. Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

    Institute of Scientific and Technical Information of China (English)

    陈长征; 夏其昌; 李伯良; 王应睐

    1997-01-01

    Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3+ ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ

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

    Science.gov (United States)

    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.

  6. Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A

    International Nuclear Information System (INIS)

    Primary cultures of rat alveolar type II cells bind radiolabeled pulmonary surfactant protein A (SP-A) with high affinity. The binding of 125I-labeled SP-A is time- and temperature-dependent and is not accompanied by significant degradation. The binding process is saturable at low concentrations of SP-A, and unlabeled SP-A readily competes with labeled SP-A for cellular binding sites. Subsequent to binding, two pools of cell-associated 125I-labeled SP-A can be identified based upon sensitivity to trypsin at 0 degree C. It is likely that the trypsin-sensitive pool comprises 125I-labeled SP-A bound to the cell surface and the trypsin-insensitive pool comprises the internalized protein. Scatchard analysis of cell surface binding of SP-A at 0.1-10 μg/ml shows positive cooperativity at concentrations between 0.1 and 1 μg/ml. Hill plots give nH = 1.34 ± 0.08 with an apparent dissociation constant K'd = 1.02 ± 0.32 μg/ml. The binding of SP-A to type II cells shows an absolute requirement for Ca2+. The putative receptor for SP-A is unaffected by treatment of type II cells with a variety of proteases and N-Glycanase. Alveolar macrophages also exhibit high-affinity binding of SP-A, but rat lung fibroblasts and the alveolar epithelial cell line L2 exhibit only nonspecific binding

  7. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes.

    Science.gov (United States)

    Van Leene, Jelle; Eeckhout, Dominique; Cannoot, Bernard; De Winne, Nancy; Persiau, Geert; Van De Slijke, Eveline; Vercruysse, Leen; Dedecker, Maarten; Verkest, Aurine; Vandepoele, Klaas; Martens, Lennart; Witters, Erwin; Gevaert, Kris; De Jaeger, Geert

    2015-01-01

    Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most advanced methods to characterize protein complexes in plants, giving a comprehensive view on the protein-protein interactions (PPIs) of a certain protein of interest (bait). The bait protein is fused to a double affinity tag, which consists of a protein G tag and a streptavidin-binding peptide separated by a very specific protease cleavage site, allowing highly specific protein complex isolation under near-physiological conditions. Implementation of this optimized TAP tag, combined with ultrasensitive MS, means that these experiments can be performed on small amounts (25 mg of total protein) of protein extracts from Arabidopsis cell suspension cultures. It is also possible to use this approach to isolate low abundant protein complexes from Arabidopsis seedlings, thus opening perspectives for the exploration of protein complexes in a plant developmental context. Next to protocols for efficient biomass generation of seedlings (∼7.5 months), we provide detailed protocols for TAP (1 d), and for sample preparation and liquid chromatography-tandem MS (LC-MS/MS; ∼5 d), either from Arabidopsis seedlings or from cell cultures. For the identification of specific co-purifying proteins, we use an extended protein database and filter against a list of nonspecific proteins on the basis of the occurrence of a co-purified protein among 543 TAP experiments. The value of the provided protocols is illustrated through numerous applications described in recent literature.

  8. A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

    Science.gov (United States)

    Enander, Karin; Dolphin, Gunnar T; Liedberg, Bo; Lundström, Ingemar; Baltzer, Lars

    2004-05-17

    A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry. PMID:15146511

  9. Reaction of Native and Denatured Brucella abortus (S19) Proteins with Antibody Using Affinity Chromatography and Immunoblotting

    OpenAIRE

    Karimi, R.; A Mostafaie; B. Tabaraie; Y. Bahrami; J. Abdolalizadeh

    2005-01-01

    Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure.In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (m...

  10. Development of an epitope tag for the gentle purification of proteins by immunoaffinity chromatography: application to epitope-tagged green fluorescent protein.

    Science.gov (United States)

    Thompson, Nancy E; Arthur, Terrance M; Burgess, Richard R

    2003-12-15

    Polyol-responsive monoclonal antibodies (mAbs) are useful tools for the gentle purification of proteins and protein complexes. These are high-affinity mAbs that release the antigen in the presence of a nonchaotropic salt and a low-molecular-weight polyhydroxylated compound (polyol). The epitope for the polyol-responsive mAb NT73, which reacts with Escherichia coli RNA polymerase, was located at the C terminus of the beta' subunit. Using recombinant DNA techniques, we have identified the epitope to be within the 13-amino-acid sequence SLAELLNAGLGGS and have developed an epitope tag that can be fused to a protein of interest for use as a purification tag. This epitope tag (designated Softag1) was fused to either the N or the C terminus of the green fluorescent protein. These tagged proteins were expressed in E. coli, and the tagged proteins were purified from the soluble fraction by a single-step immunoaffinity chromatography procedure. This approach extends the powerful technique of gentle-release immunoaffinity chromatography to many expressed proteins. PMID:14656522

  11. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    Science.gov (United States)

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants. PMID:18307162

  12. Transmembrane adaptor proteins in the high-affinity IgE receptor signaling

    Directory of Open Access Journals (Sweden)

    Petr eDraber

    2012-01-01

    Full Text Available Aggregation of the high-affinity IgE receptor (FcεRI initiates a cascade of signaling events leading to release of preformed inflammatory and allergy mediators and de novo synthesis and secretion of cytokines and other compounds. The first biochemically well defined step of this signaling cascade is tyrosine phosphorylation of the FcεRI subunits by Src family kinase Lyn, followed by recruitment and activation of Syk kinase. Activity of Syk is decisive for the formation of multicomponent signaling assemblies, the signalosomes, in the vicinity of the receptors. Formation of the signalosomes is dependent on the presence of transmembrane adaptor proteins (TRAPs. These proteins are characterized by a short extracellular domain, a single transmembrane domain and a cytoplasmic tail with various motifs serving as anchors for cytoplasmic signaling molecules. In mast cells five TRAPs have been identified (LAT, NTAL, LAX, PAG and GAPT; engagement of four of them (LAT, NTAL, LAX and PAG in FcεRI signaling has been documented. Here we discuss recent progress in the understanding of how TRAPs affect FcεRI-mediated mast cell signaling. The combined data indicate that individual TRAPs have irreplaceable roles in important signaling events such as calcium response, degranulation, cytokines production and chemotaxis.

  13. Affinity chromatography for the purification of therapeutic proteins from transgenic maize using immobilized histamine.

    Science.gov (United States)

    Platis, Dimitris; Labrou, Nikolaos E

    2008-03-01

    Plant molecular pharming is a technology that uses plants as bioreactors to produce recombinant molecules of medical and veterinary importance. In the present study, we evaluated the ability of histamine (HIM), tryptamine (TRM), phenylamine (PHEM) and tyramine (TYRM) coupled to Sepharose CL-4B via a 1,4-butanediol diglycidyl ether spacer to bind and purify human monoclonal anti-HIV antibody 2F5 (mAb 2F5) from spiked maize seed and tobacco leaf extracts. Detailed studies were carried out to determine the factors that affect the chromatographic behaviour of mAb 2F5 and also maize seed and tobacco leaf proteins. All affinity adsorbents showed a reduced capacity to bind and a reduced ability to purify proteins from tobacco extract compared to maize extract. Under optimal conditions, HIM exhibited high selectivity for mAb 2F5 and allowed a high degree of purification (>95% purity) and recovery (>90%) in a single step with salt elution (0.4 M KCl) from spiked maize seed extract. Analysis of the purified antibody fraction by ELISA and Western blot showed that the antibody was fully active and free of degraded variants or modified forms. The efficacy of the system was assessed further using a second therapeutic antibody (human monoclonal anti-HIV antibody mAb 2G12) and a therapeutic enzyme (alpha-chymotrypsin). HIM may find application in the purification of a wide range of biopharmaceuticals from transgenic plants.

  14. Control of Protein Affinity of Bioactive Nanocellulose and Passivation Using Engineered Block and Random Copolymers.

    Science.gov (United States)

    Vuoriluoto, Maija; Orelma, Hannes; Zhu, Baolei; Johansson, Leena-Sisko; Rojas, Orlando J

    2016-03-01

    We passivated TEMPO-oxidized cellulose nanofibrils (TOCNF) toward human immunoglobulin G (hIgG) by modification with block and random copolymers of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA). The block copolymers reversibly adsorbed on TOCNF and were highly effective in preventing nonspecific interactions with hIgG, especially if short PDMAEMA blocks were used. In such cases, total protein rejection was achieved. This is in contrast to typical blocking agents, which performed poorly. When an anti-human IgG biointerface was installed onto the passivated TOCNF, remarkably high affinity antibody-antigen interactions were observed (0.90 ± 0.09 mg/m(2)). This is in contrast to the nonpassivated biointerface, which resulted in a significant false response. In addition, regeneration of the biointerface was possible by low pH aqueous wash. Protein A from Staphylococcus aureus was also utilized to successfully increase the sensitivity for human IgG recognition (1.28 ± 0.11 mg/m(2)). Overall, the developed system based on TOCNF modified with multifunctional polymers can be easily deployed as bioactive material with minimum fouling and excellent selectivity. PMID:26844956

  15. Affinity Association Between Polynucleotide, Glycoprotein, or Sulfated Polysaccharides and Disease-Associated Prion Protein

    Directory of Open Access Journals (Sweden)

    Kazuo Tsukui

    2009-01-01

    Full Text Available Proteinase-K resistant prion protein (PrPres has the property to aggregate in TSE-injured animal tissues. We have developed a test method to discriminate scrapie-infected and mock-infected hamsters by detecting the PrPres in plasma. It seemed that aggregation of the PrPres with some heterogeneous molecule(s enabled successful detection by this method. In order to investigate which molecule became the partner in the PrPres aggregates; we examined some molecules that could presumably have this ability. As a result, we found synthetic Poly-A RNA, especially in its denatured form, to be the most effective entity although glycoprotein, sulfated polysaccharide showed less effectiveness. DNA in the denatured form also has a high affinity, although in the presence of protein the effectiveness unsuccessful. On the basis of this result, it is possible that the PrPres aggregate in scrapie-infected hamster plasma is composed of PrPres and RNA.

  16. Large-scale analysis of in Vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N;

    2003-01-01

    Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poo...

  17. Renal protein reactivity and stability of antibiotic amphenicols: structure and affinity.

    Science.gov (United States)

    Ding, Fei; Peng, Wei; Peng, Yu-Kui; Jiang, Yu-Ting

    2014-10-01

    In the present work, the molecular recognition of the oldest active amphenicols by the most popular renal carrier, lysozyme, was deciphered by using fluorescence, circular dichroism (CD) and molecular modeling at the molecular scale. Steady state fluorescence data showed that the recognition of amphenicol by lysozyme yields a static type of fluorescence quenching. This corroborates time-resolved fluorescence results that lysozyme-amphenicol adduct formation has a moderate affinity of 10(4) M(-1), and the driving forces were found to be chiefly hydrogen bonds, hydrophobic interactions and π stacking. Far-UV CD spectra confirmed that the spatial structure of lysozyme was slightly changed with a distinct reduction of α-helices in the presence of amphenicol, suggesting partial destabilization of the protein. Furthermore, via the extrinsic 8-anilino-1-naphthalenesulfonic acid fluorescence spectral properties and molecular modeling, one could see that the amphenicol binding site was situated at the deep crevice on the protein surface, and the ligand was also near to several crucial amino acid residues, such as Trp-62, Trp-63 and Arg-73. Simultaneously, contrastive studies of protein-amphenicols revealed clearly that some substituting groups, e.g. nitryl in the molecular structure of ligands, may be vitally important for the recognition activity of amphenicols with lysozyme. Due to the connection of amphenicols with fatal detrimental effects and because lysozyme has been applied as a drug carrier for proximal tubular targeting, the discussion herein is necessary for rational antibiotic use, development of safe antibiotics and particularly a better appraisal of the risks associated with human exposure to toxic agrochemicals. PMID:25016933

  18. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    Science.gov (United States)

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. PMID:26938582

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

    Science.gov (United States)

    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.

  20. Engineering protein therapeutics: predictive performances of a structure-based virtual affinity maturation protocol.

    Science.gov (United States)

    Oberlin, Michael; Kroemer, Romano; Mikol, Vincent; Minoux, Hervé; Tastan, Erdogan; Baurin, Nicolas

    2012-08-27

    The implementation of a structure based virtual affinity maturation protocol and evaluation of its predictivity are presented. The in silico protocol is based on conformational sampling of the interface residues (using the Dead End Elimination/A* algorithm), followed by the estimation of the change of free energy of binding due to a point mutation, applying MM/PBSA calculations. Several implementations of the protocol have been evaluated for 173 mutations in 7 different protein complexes for which experimental data were available: the use of the Boltzamnn averaged predictor based on the free energy of binding (ΔΔG(*)) combined with the one based on its polar component only (ΔΔE(pol*)) led to the proposal of a subset of mutations out of which 45% would have successfully enhanced the binding. When focusing on those mutations that are less likely to be introduced by natural in vivo maturation methods (99 mutations with at least two base changes in the codon), the success rate is increased to 63%. In another evaluation, focusing on 56 alanine scanning mutations, the in silico protocol was able to detect 89% of the hot-spots. PMID:22788756

  1. Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

    Science.gov (United States)

    Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E

    2015-12-01

    Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

  2. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency.

    Science.gov (United States)

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua; Teissedre, Pierre-Louis

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed. PMID:27518822

  3. Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Huaying, E-mail: zhaoh3@mail.nih.gov; Schuck, Peter, E-mail: zhaoh3@mail.nih.gov [National Institutes of Health, Bethesda, MD 20892 (United States)

    2015-01-01

    Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design.

  4. Combining biophysical methods for the analysis of protein complex stoichiometry and affinity in SEDPHAT

    International Nuclear Information System (INIS)

    Global multi-method analysis for protein interactions (GMMA) can increase the precision and complexity of binding studies for the determination of the stoichiometry, affinity and cooperativity of multi-site interactions. The principles and recent developments of biophysical solution methods implemented for GMMA in the software SEDPHAT are reviewed, their complementarity in GMMA is described and a new GMMA simulation tool set in SEDPHAT is presented. Reversible macromolecular interactions are ubiquitous in signal transduction pathways, often forming dynamic multi-protein complexes with three or more components. Multivalent binding and cooperativity in these complexes are often key motifs of their biological mechanisms. Traditional solution biophysical techniques for characterizing the binding and cooperativity are very limited in the number of states that can be resolved. A global multi-method analysis (GMMA) approach has recently been introduced that can leverage the strengths and the different observables of different techniques to improve the accuracy of the resulting binding parameters and to facilitate the study of multi-component systems and multi-site interactions. Here, GMMA is described in the software SEDPHAT for the analysis of data from isothermal titration calorimetry, surface plasmon resonance or other biosensing, analytical ultracentrifugation, fluorescence anisotropy and various other spectroscopic and thermodynamic techniques. The basic principles of these techniques are reviewed and recent advances in view of their particular strengths in the context of GMMA are described. Furthermore, a new feature in SEDPHAT is introduced for the simulation of multi-method data. In combination with specific statistical tools for GMMA in SEDPHAT, simulations can be a valuable step in the experimental design

  5. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency

    Science.gov (United States)

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed. PMID:27518822

  6. In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

    Full Text Available Abstract Background Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. Results Glutathione was conjugated to low molecular weight poly(ethylene glycol diacrylate (PEGDA via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH homogenates, we were able to purify a glutathione S-transferase (GST green fluorescent protein (GFP fusion protein (GST-GFP from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. Conclusions GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

  7. Improving the scoring of protein-ligand binding affinity by including the effects of structural water and electronic polarization.

    Science.gov (United States)

    Liu, Jinfeng; He, Xiao; Zhang, John Z H

    2013-06-24

    Docking programs that use scoring functions to estimate binding affinities of small molecules to biological targets are widely applied in drug design and drug screening with partial success. But accurate and efficient scoring functions for protein-ligand binding affinity still present a grand challenge to computational chemists. In this study, the polarized protein-specific charge model (PPC) is incorporated into the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method to rescore the binding poses of some protein-ligand complexes, for which docking programs, such as Autodock, could not predict their binding modes correctly. Different sampling techniques (single minimized conformation and multiple molecular dynamics (MD) snapshots) are used to test the performance of MM/PBSA combined with the PPC model. Our results show the availability and effectiveness of this approach in correctly ranking the binding poses. More importantly, the bridging water molecules are found to play an important role in correctly determining the protein-ligand binding modes. Explicitly including these bridging water molecules in MM/PBSA calculations improves the prediction accuracy significantly. Our study sheds light on the importance of both bridging water molecules and the electronic polarization in the development of more reliable scoring functions for predicting molecular docking and protein-ligand binding affinity. PMID:23651068

  8. Fluorescence Enhancement of Fluorescein Isothiocyanate-Labeled Protein A Caused by Affinity Binding with Immunoglobulin G in Bovine Plasma

    Directory of Open Access Journals (Sweden)

    Kiyotaka Sakai

    2009-10-01

    Full Text Available Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A caused by the binding with immunoglobulin G (IgG in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 μg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

  9. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (Garcinia mangostana L).

    Science.gov (United States)

    Paengkoum, P; Phonmun, T; Liang, J B; Huang, X D; Tan, H Y; Jahromi, M F

    2015-10-01

    The objectives of this study were to determine the molecular weight of condensed tannins (CT) extracted from mangosteen (Garcinia mangostana L) peel, its protein binding affinity and effects on fermentation parameters including total gas, methane (CH4) and volatile fatty acids (VFA) production. The average molecular weight (Mw) of the purified CT was 2,081 Da with a protein binding affinity of 0.69 (the amount needed to bind half the maximum bovine serum albumin). In vitro gas production declined by 0.409, 0.121, and 0.311, respectively, while CH4 production decreased by 0.211, 0.353, and 0.549, respectively, with addition of 10, 20, and 30 mg CT/500 mg dry matter (DM) compared to the control (p<0.05). The effects of CT from mangosteen-peel on in vitro DM degradability (IVDMD) and in vitro N degradability was negative and linear (p<0.01). Total VFA, concentrations of acetic, propionic, butyric and isovaleric acids decreased linearly with increasing amount of CT. The aforementioned results show that protein binding affinity of CT from mangosteen-peel is lower than those reported for Leucaena forages, however, the former has stronger negative effect on IVDMD. Therefore, the use of mangosteen-peel as protein source and CH4 mitigating agent in ruminant feed requires further investigations. PMID:26323400

  10. Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

    Science.gov (United States)

    Kang, Yu; Gohlke, Ulrich; Engström, Olof; Hamark, Christoffer; Scheidt, Tom; Kunstmann, Sonja; Heinemann, Udo; Widmalm, Göran; Santer, Mark; Barbirz, Stefanie

    2016-07-27

    Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system. PMID:27045683

  11. The development of an affinity evaluation and prediction system by using protein–protein docking simulations and parameter tuning

    Directory of Open Access Journals (Sweden)

    Koki Tsukamoto

    2009-01-01

    Full Text Available Koki Tsukamoto1, Tatsuya Yoshikawa1,2, Kiyonobu Yokota1, Yuichiro Hourai1, Kazuhiko Fukui11Computational Biology Research Center (CBRC, National Institute of Advanced Industrial Science and Technology (AIST, Koto-ku, Tokyo, Japan; 2Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Toyonaka, Osaka, JapanAbstract: A system was developed to evaluate and predict the interaction between protein pairs by using the widely used shape complementarity search method as the algorithm for docking simulations between the proteins. We used this system, which we call the affinity evaluation and prediction (AEP system, to evaluate the interaction between 20 protein pairs. The system first executes a “round robin” shape complementarity search of the target protein group, and evaluates the interaction between the complex structures obtained by the search. These complex structures are selected by using a statistical procedure that we developed called ‘grouping’. At a prevalence of 5.0%, our AEP system predicted protein–protein interactions with a 50.0% recall, 55.6% precision, 95.5% accuracy, and an F-measure of 0.526. By optimizing the grouping process, our AEP system successfully predicted 10 protein pairs (among 20 pairs that were biologically relevant combinations. Our ultimate goal is to construct an affinity database that will provide cell biologists and drug designers with crucial information obtained using our AEP system.Keywords: protein–protein interaction, affinity analysis, protein–protein docking, FFT, massive parallel computing

  12. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins.

    Science.gov (United States)

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-04-19

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  13. Affinity-controlled protein encapsulation into sub-30 nm telodendrimer nanocarriers by multivalent and synergistic interactions.

    Science.gov (United States)

    Wang, Xu; Shi, Changying; Zhang, Li; Bodman, Alexa; Guo, Dandan; Wang, Lili; Hall, Walter A; Wilkens, Stephan; Luo, Juntao

    2016-09-01

    Novel nanocarriers are highly demanded for the delivery of heterogeneous protein therapeutics for disease treatments. Conventional nanoparticles for protein delivery are mostly based on the diffusion-limiting mechanisms, e.g., physical trapping and entanglement. We develop herein a novel linear-dendritic copolymer (named telodendrimer) nanocarrier for efficient protein delivery by affinitive coating. This affinity-controlled encapsulation strategy provides nanoformulations with a small particle size (capacity (>50% w/w) and maintained protein bioactivity. We integrate multivalent electrostatic and hydrophobic functionalities synergistically into the well-defined telodendrimer scaffold to fine-tune protein binding affinity and delivery properties. The ion strength and density of the charged groups as well as the structure of the hydrophobic segments are important and their combinations in telodendrimers are crucial for efficient protein encapsulation. We have conducted a series of studies to understand the mechanism and kinetic process of the protein loading and release, utilizing electrophoresis, isothermal titration calorimetry, Förster resonance energy transfer spectroscopy, bio-layer interferometry and computational methods. The optimized nanocarriers are able to deliver cell-impermeable therapeutic protein intracellularly to kill cancer cells efficiently. In vivo imaging studies revealed cargo proteins preferentially accumulate in subcutaneous tumors and retention of peptide therapeutics is improved in an orthotopic brain tumor, these properties are evidence of the improved pharmacokinetics and biodistributions of protein therapeutics delivered by telodendrimer nanoparticles. This study presents a bottom-up strategy to rationally design and fabricate versatile nanocarriers for encapsulation and delivery of proteins for numerous applications. PMID:27294543

  14. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Lina Lu

    2015-05-01

    Full Text Available Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed.

  15. Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it.

    Science.gov (United States)

    Bacon, J R; Rhodes, M J

    2000-03-01

    Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin. PMID:10725160

  16. Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it.

    Science.gov (United States)

    Bacon, J R; Rhodes, M J

    2000-03-01

    Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin.

  17. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus;

    2013-01-01

    in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads...... prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture...... of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein...

  18. Structures of the Ultra-High-Affinity Protein–Protein Complexes of Pyocins S2 and AP41 and Their Cognate Immunity Proteins from Pseudomonas aeruginosa

    Science.gov (United States)

    Joshi, Amar; Grinter, Rhys; Josts, Inokentijs; Chen, Sabrina; Wojdyla, Justyna A.; Lowe, Edward D.; Kaminska, Renata; Sharp, Connor; McCaughey, Laura; Roszak, Aleksander W.; Cogdell, Richard J.; Byron, Olwyn; Walker, Daniel; Kleanthous, Colin

    2015-01-01

    How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins is an ultra-high-affinity interaction that is essential for the neutralisation of endogenous DNase catalytic activity and for protection against exogenous DNase bacteriocins. The colicin DNase–Im interaction is a model system for the study of high-affinity protein–protein interactions. However, despite the fact that closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this interaction has only been studied using colicins from Escherichia coli. In this work, we present the first crystal structures of two pyocin DNase–Im complexes from Pseudomonas aeruginosa, pyocin S2 DNase–ImS2 and pyocin AP41 DNase–ImAP41. These structures represent divergent DNase–Im subfamilies and are important in extending our understanding of protein–protein interactions for this important class of high-affinity protein complex. A key finding of this work is that mutations within the immunity protein binding energy hotspot, helix III, are tolerated by complementary substitutions at the DNase–Immunity protein binding interface. Im helix III is strictly conserved in colicins where an Asp forms polar interactions with the DNase backbone. ImAP41 contains an Asp-to-Gly substitution in helix III and our structures show the role of a co-evolved substitution where Pro in DNase loop 4 occupies the volume vacated and removes the unfulfilled hydrogen bond. We observe the co-evolved mutations in other DNase–Immunity pairs that appear to underpin the split of this family into two distinct groups. PMID:26215615

  19. Structure-based rational design of a Toll-like receptor 4 (TLR4 decoy receptor with high binding affinity for a target protein.

    Directory of Open Access Journals (Sweden)

    Jieun Han

    Full Text Available Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4 decoy receptor composed of leucine-rich repeat (LRR modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2. Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (K(D one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

  20. Use of surface plasmon resonance for the measurement of low affinity binding interactions between HSP72 and measles virus nucleocapsid protein

    Directory of Open Access Journals (Sweden)

    Zhang Xinsheng

    2003-01-01

    Full Text Available The 72 kDa heat shock protein (HSP72 is a molecular chaperone that binds native protein with low affinity. These interactions can alter function of the substrate, a property known as HSP-mediated activity control. In the present work, BIAcore instrumentation was used to monitor binding reactions between HSP72 and naturally occurring sequence variants of the measles virus (MV nucleocapsid protein (N, a structural protein regulating transcription/replication of the viral genome. Binding reactions employed synthetic peptides mimicking a putative HSP72 binding motif of N. Sequences were identified that bound HSP72 with affinities comparable to well-characterized activity control reactions. These sequences, but not those binding with lesser affinity, supported HSP72 activity control of MV transcription/replication. BIAcore instrumentation thus provides an effective way to measure biologically relevant low affinity interactions with structural variants of viral proteins.

  1. Affinity purification of human factor H on polypeptides derived from streptococcal m protein: enrichment of the Y402 variant.

    Directory of Open Access Journals (Sweden)

    O Rickard Nilsson

    Full Text Available Recent studies indicate that defective activity of complement factor H (FH is associated with several human diseases, suggesting that pure FH may be used for therapy. Here, we describe a simple method to isolate human FH, based on the specific interaction between FH and the hypervariable region (HVR of certain Streptococcus pyogenes M proteins. Special interest was focused on the FH polymorphism Y402H, which is associated with the common eye disease age-related macular degeneration (AMD and has also been implicated in the binding to M protein. Using a fusion protein containing two copies of the M5-HVR, we found that the Y402 and H402 variants of FH could be efficiently purified by single-step affinity chromatography from human serum containing the corresponding protein. Different M proteins vary in their binding properties, and the M6 and M5 proteins, but not the M18 protein, showed selective binding of the FH Y402 variant. Accordingly, chromatography on a fusion protein derived from the M6-HVR allowed enrichment of the Y402 protein from serum containing both variants. Thus, the exquisite binding specificity of a bacterial protein can be exploited to develop a simple and robust procedure to purify FH and to enrich for the FH variant that protects against AMD.

  2. Enhancing Protein Capture Using a Combination of Nanoyeast Single-Chain Fragment Affinity Reagents and Alternating Current Electrohydrodynamic Forces.

    Science.gov (United States)

    Vaidyanathan, Ramanathan; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Trau, Matt

    2015-12-01

    New high-performance detection technologies and more robust protein capture agents can be combined to both rapidly and specifically capture and detect protein biomarkers associated with disease in complex biological samples. Here we demonstrate the use of recently developed recombinant affinity reagents, namely nanoyeast-scFv, in combination with alternating current electrohydrodynamic (ac-EHD)-induced shear forces, to enhance capture performance during protein biomarker analysis. The use of ac-EHD significantly improves fluid transport across the capture domain, resulting in enhanced sensor-target interaction and simultaneous displacement of nonspecific molecules from the electrode surface. We demonstrate this simple proof-of-concept approach for the capture and detection of Entamoeba histolytica antigens from disinfected stool, within a span of 5 min using an ac-EHD microfluidic device. Under an ac-EHD field, antigens were captured on a nanoyeast-scFv immobilized device and subsequently detected using a quantum dot conjugated antibody. This immunosensor specifically detected antigen in disinfected stool with low background noise at concentrations down to 58.8 fM with an interassay reproducibility (%RSD of n = 3) gold electrodes in disinfected stool. We predict this rapid and sensitive approach using these stable affinity reagents may offer a new methodology to detect protein disease biomarkers from biological matrices. PMID:26551436

  3. High performance aptamer affinity chromatography for single-step selective extraction and screening of basic protein lysozyme.

    Science.gov (United States)

    Han, Bin; Zhao, Chao; Yin, Junfa; Wang, Hailin

    2012-08-15

    A DNA aptamer based high-performance affinity chromatography is developed for selective extraction and screening of a basic protein lysozyme. First, a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic column was synthesized in situ by thermally initiated radical polymerization, and then an anti-lysozyme DNA aptamer was covalently immobilized on the surface of the monolith through a 16-atom spacer arm. The target protein lysozyme but non-target proteins can be trapped by the immobilized anti-lysozyme DNA aptamer. In contrast, lysozyme cannot be trapped by the immobilized oligodeoxynucleotide that does not contain the sequence of the anti-lysozyme DNA aptamer. The study clearly demonstrates the trapping of lysozyme by the immobilized anti-lysozyme DNA aptamer is mainly due to specific recognition rather than simple electrostatic interaction of positively charged protein and the negatively charged DNA. The inter-day precision was determined as 0.8% for migration time and 4.2% for peak area, respectively. By the use of aptamer affinity monolith, a screening strategy is developed to selectively extract lysozyme from chicken egg white, showing the advantages of high efficiency, low cost and ease-of-operation.

  4. Magneto-nanosensor platform for probing low-affinity protein-protein interactions and identification of a low-affinity PD-L1/PD-L2 interaction.

    Science.gov (United States)

    Lee, Jung-Rok; Bechstein, Daniel J B; Ooi, Chin Chun; Patel, Ashka; Gaster, Richard S; Ng, Elaine; Gonzalez, Lino C; Wang, Shan X

    2016-01-01

    Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1-PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2. PMID:27447090

  5. Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

    Directory of Open Access Journals (Sweden)

    Utut Widyastuti Suharsono

    2008-11-01

    Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

  6. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (Garcinia mangostana L).

    Science.gov (United States)

    Paengkoum, P; Phonmun, T; Liang, J B; Huang, X D; Tan, H Y; Jahromi, M F

    2015-10-01

    The objectives of this study were to determine the molecular weight of condensed tannins (CT) extracted from mangosteen (Garcinia mangostana L) peel, its protein binding affinity and effects on fermentation parameters including total gas, methane (CH4) and volatile fatty acids (VFA) production. The average molecular weight (Mw) of the purified CT was 2,081 Da with a protein binding affinity of 0.69 (the amount needed to bind half the maximum bovine serum albumin). In vitro gas production declined by 0.409, 0.121, and 0.311, respectively, while CH4 production decreased by 0.211, 0.353, and 0.549, respectively, with addition of 10, 20, and 30 mg CT/500 mg dry matter (DM) compared to the control (pmangosteen-peel on in vitro DM degradability (IVDMD) and in vitro N degradability was negative and linear (pmangosteen-peel is lower than those reported for Leucaena forages, however, the former has stronger negative effect on IVDMD. Therefore, the use of mangosteen-peel as protein source and CH4 mitigating agent in ruminant feed requires further investigations.

  7. Polymerization Degrees, Molecular Weights and Protein-Binding Affinities of Condensed Tannin Fractions from a Leucaena leucocephala Hybrid

    Directory of Open Access Journals (Sweden)

    Mookiah Saminathan

    2014-06-01

    Full Text Available Condensed tannins (CTs form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1–F5 measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight—from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

  8. Heme-binding plasma membrane proteins of K562 erythroleukemia cells: Adsorption to heme-microbeads, isolation with affinity chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Majuri, R. (Minerva Foundation Institute for Medical Research, Helsinki (Finland))

    1989-01-01

    Heme-microbeads attached themselves to the surface of viable K562 cells in a manner inhibitable by free hemin, indicating heme-recptor interaction. The microbeads were at first evenly distributed, but after prolonged incubation at 37 deg. C they formed a cap on one pole of the cells indicating clustering of the membrane heme receptors. Membrane proteins were labeled by culturing the cells in the presence of {sup 35}S-methionine and were then solubilized with Triton X-114. The hydrophobic proteins contained about 20% of the total bound label. The solubilized membrane proteins were subsequently adsorbed to a heme-Sepharose affinity gel. According to SDS-electrophorsis and subsequent autoradiography, the immobilized heme captures two proteins or a protein with two polypeptides of 20 000 and 32 000 daltons. The larger of these was only wekly labeled with {sup 35}S. The same two bands were observed if the cell surface proteins were labeled with {sup 125}I by the lactoperoxidase method and the subsequently solubilized membrane proteins were isolated with heme-Sepharose. (author).

  9. UO₂²⁺ uptake by proteins: understanding the binding features of the super uranyl binding protein and design of a protein with higher affinity.

    Science.gov (United States)

    Odoh, Samuel O; Bondarevsky, Gary D; Karpus, Jason; Cui, Qiang; He, Chuan; Spezia, Riccardo; Gagliardi, Laura

    2014-12-17

    The capture of uranyl, UO2(2+), by a recently engineered protein (Zhou et al. Nat. Chem. 2014, 6, 236) with high selectivity and femtomolar sensitivity has been examined by a combination of density functional theory, molecular dynamics, and free-energy simulations. It was found that UO2(2+) is coordinated to five carboxylate oxygen atoms from four amino acid residues of the super uranyl binding protein (SUP). A network of hydrogen bonds between the amino acid residues coordinated to UO2(2+) and residues in its second coordination sphere also affects the protein's uranyl binding affinity. Free-energy simulations show how UO2(2+) capture is governed by the nature of the amino acid residues in the binding site, the integrity and strength of the second-sphere hydrogen bond network, and the number of water molecules in the first coordination sphere. Alteration of any of these three factors through mutations generally results in a reduction of the binding free energy of UO2(2+) to the aqueous protein as well as of the difference between the binding free energies of UO2(2+) and other ions (Ca(2+), Cu(2+), Mg(2+), and Zn(2+)), a proxy for the protein's selectivity over these ions. The results of our free-energy simulations confirmed the previously reported experimental results and allowed us to discover a mutant of SUP, specifically the GLU64ASP mutant, that not only binds UO2(2+) more strongly than SUP but that is also more selective for UO2(2+) over other ions. The predictions from the computations were confirmed experimentally.

  10. Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein

    International Nuclear Information System (INIS)

    The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides. Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion. However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage. To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14. Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14. Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts. Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography. The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction. We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells

  11. Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: application to human cardiac troponin I.

    Science.gov (United States)

    Dalluge, J J; Sander, L C

    1998-12-15

    An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (approximately 5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femto-molar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed. PMID:9868922

  12. Alcohol Binding to the Odorant Binding Protein LUSH: Multiple Factors Affecting Binding Affinities

    OpenAIRE

    Ader, Lauren; Jones, David N. M.; Lin, Hai

    2010-01-01

    Density function theory (DFT) calculations have been carried out to investigate the binding of alcohols to the odorant binding protein LUSH from Drosophila melanogaster. LUSH is one of the few proteins known to bind to ethanol at physiologically relevant concentrations and where high-resolution structural information is available for the protein bound to alcohol at these concentrations. The structures of the LUSH–alcohol complexes identify a set of specific hydrogen-bonding interactions as cr...

  13. Affinity tagging & purification of the fucose binding LecB protein

    OpenAIRE

    Creavin, Aileen; O'Connor, Brendan

    2007-01-01

    The fucose binding LecB protein is one of two identified lectins produced by the opportunistic pathogen Pseudomonas aeruginosa (PA01) and is implicated in contributing to its virulence. A large number of homologous proteins have been identified in other bacterial species that exhibit extremely high sequence identity and similarity to LecB. However, key amino acid residues known to participate in fucose binding in LecB are altered in many of these proteins. Some of these proteins have been sho...

  14. Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins.

    Science.gov (United States)

    Xu, Jingjing; Ambrosini, Serena; Tamahkar, Emel; Rossi, Claire; Haupt, Karsten; Tse Sum Bui, Bernadette

    2016-01-11

    We describe a potentially universal, simple and cheap method to prepare water-compatible molecularly imprinted polymer nanoparticles (MIP-NPs) as synthetic antibodies against proteins. The strategy is based on a solid phase synthesis approach where glass beads (GBs) are functionalized with a metal chelate, acting as a general affinity ligand to attract surface-bound histidines present on proteins. This configuration enables an oriented immobilization of the proteins, upon which thermoresponsive MIP-NPs are synthesized. The GBs play the role of both a reactor and a separation column since, after synthesis, the MIP-NPs are released from the support by a simple temperature change, resulting in protein-free polymers. The resulting MIP-NPs are endowed with improved binding site homogeneity, since the binding sites have the same orientation. Moreover, they are stable (no aggregation) in a buffer solution for prolonged storage time and exhibit apparent dissociation constants in the nanomolar range, with little or no cross-reactivity toward other proteins. PMID:26644006

  15. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  16. Dissecting the Binding Mode of Low Affinity Phage Display Peptide Ligands to Protein Targets by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Ming, Shonoi A;

    2014-01-01

    of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize interactions of low affinity peptides with their cognate protein targets. The HDX-MS workflow was optimized to accurately detect low-affinity peptide-protein interactions by use of ion mobility, electron transfer dissociation, non...... of KDM4C, indicating distinct binding modes. In contrast, the perturbation site of another PD-selected peptide inhibiting the function of KDM1A maps to a GST-tag. Our results demonstrate that HDX-MS can validate and map weak peptide-protein interactions, and pave the way for understanding and optimizing...

  17. Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins : a wash step with a low concentration of EDTA

    NARCIS (Netherlands)

    Westra, DF; Welling, GW; Koedijk, DGAM; Scheffer, AJ; The, TH; Welling-Wester, S

    2001-01-01

    Immobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for t

  18. Design, synthesis, and application of a hydrazide-functionalized isotope-coded affinity tag for the quantification of oxylipid-protein conjugates.

    Science.gov (United States)

    Han, Bingnan; Stevens, Jan F; Maier, Claudia S

    2007-05-01

    An isotopically coded affinity probe was developed and evaluated for the characterization and quantification of proteins adducted by 2-alkenals derived from lipid peroxidation (LPO) processes. Lipid-derived 2-alkenals, such as acrolein and 4-hydroxy-2-nonenal (HNE), have the ability to react with cysteine, histidine, and lysine residues in proteins, thus causing protein damage and loss of protein function. Such modifications of proteins are difficult to characterize in biological samples by mass spectrometry due to the complexity of protein extracts and the low abundance of adducted proteins. The novel aldehyde-reactive, hydrazide-functionalized, isotope-coded affinity tag (HICAT) described in this study was found effective for the selective isolation, detection, and quantification of Michael-type adducts of 2-alkenals with proteins using a combination of affinity isolation, nanoLC, and matrix-assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS). The chemical and mass spectrometric properties of the new probe are demonstrated on a model protein treated with HNE. The efficacy of HICAT for the analysis of complex samples was tested using preparations of mitochondrial proteins that were modified in vitro with HNE. The potential of the HICAT strategy for the identification, characterization, and quantification of in vivo oxylipid-protein conjugates is demonstrated on cardiac mitochondrial protein preparations, in which, for example, the ADP/ATP translocase 1 was found adducted to the 2-alkenals, acrolein and 4-hydroxy-2-hexenal, at Cys-256.

  19. HADDOCK2P2I : A biophysical model for predicting the binding affinity of protein-protein interaction inhibitors

    NARCIS (Netherlands)

    Kastritis, Panagiotis L.; Garcia Lopes Maia Rodrigues, João; Bonvin, Alexandre M J J

    2014-01-01

    The HADDOCK score, a scoring function for both protein-protein and protein-nucleic acid modeling, has been successful in selecting near-native docking poses in a variety of cases, including those of the CAPRI blind prediction experiment. However, it has yet to be optimized for small molecules, and i

  20. A simple detection method for low-affinity membrane protein interactions by baculoviral display.

    Directory of Open Access Journals (Sweden)

    Toshiko Sakihama

    Full Text Available BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV. In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA. Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L, and glucocorticoid-induced TNFR family-related protein (GITR-GITR ligand (GITRL. Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

  1. Comparing the Affinity of GTPase-binding Proteins using Competition Assays.

    Science.gov (United States)

    Williamson, Rosalind C; Bass, Mark D

    2015-01-01

    In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.

  2. Diversity selection of compounds based on 'protein affinity fingerprints' improves sampling of bioactive chemical space.

    Science.gov (United States)

    Nguyen, Ha P; Koutsoukas, Alexios; Mohd Fauzi, Fazlin; Drakakis, Georgios; Maciejewski, Mateusz; Glen, Robert C; Bender, Andreas

    2013-09-01

    Diversity selection is a frequently applied strategy for assembling high-throughput screening libraries, making the assumption that a diverse compound set increases chances of finding bioactive molecules. Based on previous work on experimental 'affinity fingerprints', in this study, a novel diversity selection method is benchmarked that utilizes predicted bioactivity profiles as descriptors. Compounds were selected based on their predicted activity against half of the targets (training set), and diversity was assessed based on coverage of the remaining (test set) targets. Simultaneously, fingerprint-based diversity selection was performed. An original version of the method exhibited on average 5% and an improved version on average 10% increase in target space coverage compared with the fingerprint-based methods. As a typical case, bioactivity-based selection of 231 compounds (2%) from a particular data set ('Cutoff-40') resulted in 47.0% and 50.1% coverage, while fingerprint-based selection only achieved 38.4% target coverage for the same subset size. In conclusion, the novel bioactivity-based selection method outperformed the fingerprint-based method in sampling bioactive chemical space on the data sets considered. The structures retrieved were structurally more acceptable to medicinal chemists while at the same time being more lipophilic, hence bioactivity-based diversity selection of compounds would best be combined with physicochemical property filters in practice.

  3. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    International Nuclear Information System (INIS)

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1, Val1, Asn2, Gln3, His4, Ser8, His9, Glu12, Tyr15, Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3, Ala4] IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15, Leu16] IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, [Gln3, Ala4, Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I

  4. Combining metal oxide affinity chromatography (MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

    Directory of Open Access Journals (Sweden)

    Weckwerth Wolfram

    2005-11-01

    Full Text Available Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC and selective ion trap mass spectrometry. The complete approach involves (i enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment, (iii identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to

  5. A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

    Directory of Open Access Journals (Sweden)

    Giordanni C. Dantas

    2016-06-01

    Full Text Available Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac, is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

  6. A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri.

    Science.gov (United States)

    Dantas, Giordanni C; Martins, Paula M M; Martins, Daniela A B; Gomes, Eleni; Ferreira, Henrique

    2016-01-01

    Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo. PMID:26991273

  7. Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water

    Energy Technology Data Exchange (ETDEWEB)

    Dalvit, Claudio; Pevarello, Paolo; Tato, Marco; Veronesi, Marina; Vulpetti, Anna; Sundstroem, Michael [Pharmacia (Italy)

    2000-09-15

    A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H{sub 2}O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented.

  8. Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein-ligand interactions based on a study on caspase 3 inhibitors.

    Science.gov (United States)

    Erdas, Ozlem; Andac, Cenk A; Gurkan-Alp, A Selen; Alpaslan, Ferda Nur; Buyukbingol, Erdem

    2015-01-01

    The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein-ligand complexes. CIFAP-2 method is established based on a protein-ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein-ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy ınference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein-ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3-ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC(50)) of the CASP3 inhibitors. PMID:25578823

  9. A comparison of protein kinases inhibitor screening methods using both enzymatic activity and binding affinity determination

    DEFF Research Database (Denmark)

    Rudolf, Amalie Frederikke; Skovgaard, Tine; Knapp, Stefan;

    2014-01-01

    Binding assays are increasingly used as a screening method for protein kinase inhibitors; however, as yet only a weak correlation with enzymatic activity-based assays has been demonstrated. We show that the correlation between the two types of assays can be improved using more precise screening...

  10. Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions

    DEFF Research Database (Denmark)

    Jönsson, Peter; Southcombe, Jennifer H; Santos, Ana Mafalda;

    2016-01-01

    value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with...

  11. Conformational plasticity of IgG during protein A affinity chromatography.

    Science.gov (United States)

    Gagnon, Pete; Nian, Rui

    2016-02-12

    Single step elution of a protein A column with 100mM acetate pH 3.5 produced a curvilinear gradient with pH dropping steeply at first then more gradually as it approached endpoint. IgG with a native hydrodynamic diameter of 11.5 nm began to elute at pH 6.0 with a size of 9.4 nm. IgG size continued to decrease across the peak, reaching a minimum of 2.2 nm at pH 3.9. Secondary structure of early eluting IgG was only mildly affected but later eluting fractions became increasingly non-native with the 2.2 nm population exhibiting the highest proportion of β-sheet and lowest random coil of all conformations. Size reduction and structural change of IgG through this portion of the elution peak were attributed dominantly to a pre-existing tendency of highly concentrated IgG to adopt reduced size conformations at low pH and conductivity, facilitated by the known conformational relaxation of IgG by its interaction with protein A. IgG size increased to 10.4 nm as elution pH approached 3.5 across the tailing fractions. Major loss of β-sheet and increase of α-helix and random coil were observed in parallel. Late elution of this population was attributed to it being eluted from interactions with 2 distinct protein A domains, one bound to each side of the Fc region, creating a higher dissociation constant than single-site Fc-protein A interactions, and requiring more severely disruptive conditions for elution. The high degree of conformational disruption was attributed to simultaneous interaction of both heavy chains with protein A. PMID:26805601

  12. The human homolog of Escherichia coli endonuclease V is a nucleolar protein with affinity for branched DNA structures.

    Directory of Open Access Journals (Sweden)

    Cathrine Fladeby

    Full Text Available Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV, encoded by the nfi gene, which cleaves the second phosphodiester bond 3' of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV, many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3'-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

  13. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

    Science.gov (United States)

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

    2016-05-15

    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  14. Yeast surface display for directed evolution of protein expression, affinity, and stability.

    Science.gov (United States)

    Boder, E T; Wittrup, K D

    2000-01-01

    The described protocols enable thorough screening of polypeptide libraries with high confidence in the isolation of improved clones. It should be emphasized that the protocols have been fashioned for thoroughness, rather than speed. With library plasmid DNA in hand, the time to plated candidate yeast display mutants is typically 2-3 weeks. Each of the experimental approaches required for this method is fairly standard: yeast culture, immunofluorescent labeling, flow cytometry. Protocols that are more rapid could conceivably be developed by using solid substrate separations with magnetic beads, for instance. However, loss of the two-color normalization possible with flow cytometry would remove the quantitative advantage of the method. Yeast display complements existing polypeptide library methods and opens the possibility of examining extracellular eukaryotic proteins, an important class of proteins not generally amenable to yeast two-hybrid or phage display methodologies.

  15. Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

    Science.gov (United States)

    Cole, Daniel J; Rajendra, Eeson; Roberts-Thomson, Meredith; Hardwick, Bryn; McKenzie, Grahame J; Payne, Mike C; Venkitaraman, Ashok R; Skylaris, Chris-Kriton

    2011-07-01

    The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the

  16. Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

    Directory of Open Access Journals (Sweden)

    Daniel J Cole

    2011-07-01

    Full Text Available The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and

  17. High affinity human antibody fragments to dengue virus non-structural protein 3.

    Directory of Open Access Journals (Sweden)

    Nicole J Moreland

    Full Text Available BACKGROUND: The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3 are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5' triphosphatase domain which forms the remainder of the 618-aa long protein. METHODOLOGY/PRINCIPAL FINDINGS: In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531 within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells. CONCLUSIONS/SIGNIFICANCE: Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

  18. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    Science.gov (United States)

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  19. Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation

    OpenAIRE

    Wood David W; Fong Baley A

    2010-01-01

    Abstract Background Elastin-like polypeptides (ELPs) are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up. Results In this work, we describe the high cell-density expression of self-cleaving ...

  20. Gateway synthesis of daphnane congeners and their protein kinase C affinities and cell-growth activities

    Science.gov (United States)

    Wender, Paul A.; Buschmann, Nicole; Cardin, Nathan B.; Jones, Lisa R.; Kan, Cindy; Kee, Jung-Min; Kowalski, John A.; Longcore, Kate E.

    2011-08-01

    The daphnane diterpene orthoesters constitute a structurally fascinating family of natural products that exhibit a remarkable range of potent biological activities. Although partial activity information is available for some natural daphnanes, little information exists for non-natural congeners or on how changes in structure affect mode of action, function, potency or selectivity. A gateway strategy designed to provide general synthetic access to natural and non-natural daphnanes is described and utilized in the synthesis of two novel members of this class. In this study, a commercially available tartrate derivative was elaborated through a key late-stage diversification intermediate into B-ring yuanhuapin analogues to initiate exploration of the structure-function relationships of this class. Protein kinase C was identified as a cellular target for these agents, and their activity against human lung and leukaemia cell lines was evaluated. The natural product and a novel non-natural analogue exhibited significant potency, but the epimeric epoxide was essentially inactive.

  1. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    Science.gov (United States)

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  2. The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells

    DEFF Research Database (Denmark)

    Honoré, B; Vorum, H

    2000-01-01

    in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca...

  3. An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography

    Directory of Open Access Journals (Sweden)

    Büssow Konrad

    2003-07-01

    Full Text Available Abstract Background Functional Genomics, the systematic characterisation of the functions of an organism's genes, includes the study of the gene products, the proteins. Such studies require methods to express and purify these proteins in a parallel, time and cost effective manner. Results We developed a method for parallel expression and purification of recombinant proteins with a hexahistidine tag (His-tag or glutathione S-transferase (GST-tag from bacterial expression systems. Proteins are expressed in 96-well microplates and are purified by a fully automated procedure on a pipetting robot. Up to 90 microgram purified protein can be obtained from 1 ml microplate cultures. The procedure is readily reproducible and 96 proteins can be purified in approximately three hours. It avoids clearing of crude cellular lysates and the use of magnetic affinity beads and is therefore less expensive than comparable commercial systems. We have used this method to compare purification of a set of human proteins via His-tag or GST-tag. Proteins were expressed as fusions to an N-terminal tandem His- and GST-tag and were purified by metal chelating or glutathione affinity chromatography. The purity of the obtained protein samples was similar, yet His-tag purification resulted in higher yields for some proteins. Conclusion A fully automated, robust and cost effective method was developed for the purification of proteins that can be used to quickly characterise expression clones in high throughput and to produce large numbers of proteins for functional studies. His-tag affinity purification was found to be more efficient than purification via GST-tag for some proteins.

  4. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    Science.gov (United States)

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  5. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    Science.gov (United States)

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  6. Development and Characterization of Protective Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin and Comparison with Other Subunit and Commercial Vaccines ▿

    OpenAIRE

    Frandoloso, Rafael; Martínez, Sonia; Rodríguez-Ferri, Elías F.; García-Iglesias, María José; Pérez-Martínez, Claudia; Martínez-Fernández, Beatriz; Gutiérrez-Martín, César B.

    2010-01-01

    Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was poten...

  7. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    Science.gov (United States)

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  8. A Versatile Platform to Analyze Low-Affinity and Transient Protein-Protein Interactions in Living Cells in Real Time

    Directory of Open Access Journals (Sweden)

    Yao-Cheng Li

    2014-12-01

    Full Text Available Protein-protein interactions (PPIs play central roles in orchestrating biological processes. While some PPIs are stable, many important ones are transient and hard to detect with conventional approaches. We developed ReBiL, a recombinase enhanced bimolecular luciferase complementation platform, to enable detection of weak PPIs in living cells. ReBiL readily identified challenging transient interactions between an E3 ubiquitin ligase and an E2 ubiquitin-conjugating enzyme. ReBiL’s ability to rapidly interrogate PPIs in diverse conditions revealed that some stapled α-helical peptides, a class of PPI antagonists, induce target-independent cytosolic leakage and cytotoxicity that is antagonized by serum. These results explain the requirement for serum-free conditions to detect stapled peptide activity, and define a required parameter to evaluate for peptide antagonist approaches. ReBiL’s ability to expedite PPI analysis, assess target specificity and cell permeability, and reveal off-target effects of PPI modifiers should facilitate the development of effective, cell-permeable PPI therapeutics and the elaboration of diverse biological mechanisms.

  9. The Dipole Potential Modifies the Clustering and Ligand Binding Affinity of ErbB Proteins and Their Signaling Efficiency

    Science.gov (United States)

    Kovács, Tamás; Batta, Gyula; Hajdu, Tímea; Szabó, Ágnes; Váradi, Tímea; Zákány, Florina; Csomós, István; Szöllősi, János; Nagy, Peter

    2016-01-01

    Although activation of the ErbB family of receptor tyrosine kinases (ErbB1-4) is driven by oligomerization mediated by intermolecular interactions between the extracellular, the kinase and the transmembrane domains, the transmembrane domain has been largely neglected in this regard. The largest contributor to the intramembrane electric field, the dipole potential, alters the conformation of transmembrane peptides, but its effect on ErbB proteins is unknown. Here, we show by Förster resonance energy transfer (FRET) and number and brightness (N&B) experiments that the epidermal growth factor (EGF)-induced increase in the homoassociation of ErbB1 and ErbB2 and their heteroassociation are augmented by increasing the dipole potential. These effects were even more pronounced for ErbB2 harboring an activating Val → Glu mutation in the transmembrane domain (NeuT). The signaling capacity of ErbB1 and ErbB2 was also correlated with the dipole potential. Since the dipole potential decreased the affinity of EGF to ErbB1, the augmented growth factor-induced effects at an elevated dipole potential were actually induced at lower receptor occupancy. We conclude that the dipole potential plays a permissive role in the clustering of ErbB receptors and that the effects of lipid rafts on ligand binding and receptor signaling can be partially attributed to the dipole potential. PMID:27775011

  10. Affinity of anticancer drug, daunomycin, to core histones in solution:comparison of free and cross-linked proteins

    Institute of Scientific and Technical Information of China (English)

    Azra RABBANI; Sayeh ABDOSAMADI; Naghmeh SARI-SARAF

    2007-01-01

    Aim: The interaction of anthracyclinc anticancer drugs with chromatin, nuclco-somes and historic H1 has been extensively studied. In the present study, for the first time, we have investigated the binding of anthracycline antibiotic, daunomycin,to free and cross-linked thymus core histones (CL-core) in solution and in the absence of DNA. Methods: Fluorescence, UV/Vis spectroscopy and equilibrium dialysis techniques were used. Results: The UV spectroscopy results show that daunomycin induces hypochromicity in the absorption spectra of the core histones.Fluorescence emission intensity is decreased upon daunomycin binding and the process is concentration dependent. The equilibrium dialysis shows that the bind-ing is positive cooperative with the binding sites as Scatchard plot and Hill Coef-ficient confirm it. Conclusion: The results suggest that daunomycin shows much higher affinity to core histories free in solution than to CL-core, implying that the binding is most likely due to the accessibility of these proteins to the environment.It is suggested that daunomycin binds strongly to open state of histones, such as in tumor cells, rather than to their compact structure seen in normal chromatin.

  11. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a concentration-depende

  12. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    Science.gov (United States)

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  13. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    Science.gov (United States)

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1).

  14. Data on the identification of protein interactors with the Evening Complex and PCH1 in Arabidopsis using tandem affinity purification and mass spectrometry (TAP-MS).

    Science.gov (United States)

    Huang, He; Alvarez, Sophie; Nusinow, Dmitri A

    2016-09-01

    Tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis is a powerful biochemical approach to identify protein-protein associations. Here we describe two datasets generated by a series of TAP-MS analyses to co-purify proteins associated with either ELF3 or ELF4 of the Evening Complex (EC) ("Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry" (Huang et al., 2016a) [1]) or proteins associated with PCH1, which is a newly identified output of the circadian clock to regulate photoperiodic growth in Arabidopsis thaliana ("PCH1 integrates circadian and light-signaling pathways to control photoperiod-responsive growth in Arabidopsis" (Huang et al. 2016b) [2]). We used either ELF3, ELF4 or PCH1 fused to a C-terminal tandem affinity tag (6xHis-3xFLAG) as baits and conducted purifications in various genetic mutant backgrounds. These data are discussed in recent publications [1,2], and are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002606 (for EC) and PRIDE: PXD003352 (for PCH1). PMID:27274533

  15. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-Cβ antibody

    International Nuclear Information System (INIS)

    Highlights: ► A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. ► TCR-Fc protein immobilized by an anti-Cβ antibody bound to a p/MHC tetramer. ► Binding affinity of TCR-Fc was markedly increased by binding with anti-Cβ antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100–200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cβ antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10−5 M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cβ antibody, its binding affinity for p/MHC increased by 5-fold (2.2 × 10−6 M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-Cβ antibody, which is probably due to the stabilization of the Vα/Vβ region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing

  16. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  17. Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bayne, M.L.; Applebaum, J.; Chicchi, G.G.; Hayes, N.S.; Green, B.G.; Cascieri, M.A.

    1988-05-05

    Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. (Phe/sup -1/, Val/sup 1/, Asn/sup 2/, Gln/sup 3/, His/sup 4/, Ser/sup 8/, His/sup 9/, Glu/sup 12/, Tyr/sup 15/, Leu/sup 16/)IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has >1000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. (Gln/sup 3/, Ala/sup 4/) IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. (Tyr/sup 15/, Leu/sup 16/) IGH-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. The peptide in which these four-point mutations are combined, (Gln/sup 3/, Ala/sup 4/, Tyr/sup 15/,Leu/sup 16/)IGF-I, has 600-fold reduced affinity for the serum binding proteins. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, These peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.

  18. Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

    Science.gov (United States)

    Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

    2016-07-29

    We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity.

  19. Ampicillin/penicillin-binding protein interactions as a model drug-target system to optimize affinity pull-down and mass spectrometric strategies for target and pathway identification.

    Science.gov (United States)

    von Rechenberg, Moritz; Blake, Brian Kelly; Ho, Yew-Seng J; Zhen, Yuejun; Chepanoske, Cindy Lou; Richardson, Bonnie E; Xu, Nafei; Kery, Vladimir

    2005-05-01

    The identification and validation of the targets of active compounds identified in cell-based assays is an important step in preclinical drug development. New analytical approaches that combine drug affinity pull-down assays with mass spectrometry (MS) could lead to the identification of new targets and druggable pathways. In this work, we investigate a drug-target system consisting of ampicillin- and penicillin-binding proteins (PBPs) to evaluate and compare different amino-reactive resins for the immobilization of the affinity compound and mass spectrometric methods to identify proteins from drug affinity pull-down assays. First, ampicillin was immobilized onto various amino-reactive resins, which were compared in the ampicillin-PBP model with respect to their nonspecific binding of proteins from an Escherichia coli membrane extract. Dynal M-270 magnetic beads were chosen to further study the system as a model for capturing and identifying the targets of ampicillin, PBPs that were specifically and covalently bound to the immobilized ampicillin. The PBPs were identified, after in situ digestion of proteins bound to ampicillin directly on the beads, by using either one-dimensional (1-D) or two-dimensional (2-D) liquid chromatography (LC) separation techniques followed by tandem mass spectrometry (MS/MS) analysis. Alternatively, an elution with N-lauroylsarcosine (sarcosyl) from the ampicillin beads followed by in situ digestion and 2-D LC-MS/MS analysis identified proteins potentially interacting noncovalently with the PBPs or the ampicillin. The in situ approach required only little time, resources, and sample for the analysis. The combination of drug affinity pull-down assays with in situ digestion and 2-D LC-MS/MS analysis is a useful tool in obtaining complex information about a primary drug target as well as its protein interactors. PMID:15761956

  20. Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry.

    Science.gov (United States)

    Wu, Fang; Wang, Peng; Young, Leah C; Lai, Raymond; Li, Liang

    2009-02-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), an oncogenic fusion gene protein that is characteristically found in a subset of anaplastic large cell lymphomas, promotes tumorigenesis through its functional and physical interactions with various biologically important proteins. The identification of these interacting proteins has proven to be useful to further our understanding of NPM-ALK-mediated tumorigenesis. For the first time, we performed a proteome-wide identification of NPM-ALK-binding proteins using tandem affinity purification and a highly sensitive mass spectrometric technique. Tandem affinity purification is a recently developed method that carries a lower background and higher sensitivity compared with the conventional immunoprecipitation-based protein purification protocols. The NPM-ALK gene was cloned into an HB-tagged vector and expressed in GP293 cells. Three independent experiments were performed and the reproducibility of the data was 68%. The vast majority of the previously reported NPM-ALK-binding proteins were detected. We also identified proteins that are involved in various cellular processes that were not previously described in association with NPM-ALK, such as MCM6 and MSH2 (DNA repair), Nup98 and importin 8 (subcellular protein transport), Stim1 (calcium signaling), 82Fip (RNA regulation), and BAG2 (proteosome degradation). We believe that these data highlight the functional diversity of NPM-ALK and provide new research directions for the study of the biology of this oncoprotein.

  1. Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.

    Science.gov (United States)

    Jiao, Chensong; Summerlin, Matthew; Bruzik, Karol S; Hanakahi, Leslyn

    2015-10-20

    Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems. PMID:26397942

  2. Hepatitis C virus expressing flag-tagged envelope protein 2 has unaltered infectivity and density, is specifically neutralized by flag antibodies and can be purified by affinity chromatography

    DEFF Research Database (Denmark)

    Prentø, Jannick Cornelius; Bukh, Jens

    2011-01-01

    Hepatitis C virus (HCV) purification by ultracentrifugation is difficult because of the low and heterogeneous density of native and cultured viruses. It was recently shown that inserting flag tag into envelope protein 2 (E2) of HCV permitted virus purification by affinity chromatography. However,...... physiochemical properties of flag-tagged HCV is an important improvement for utilizing these viruses for imaging, virion composition analysis and possibly vaccine development....

  3. Cyclic Lactam Hybrid α-MSH/Agouti-Related Protein (AGRP) Analogues with Nanomolar Range Binding Affinities at the Human Melanocortin Receptors

    OpenAIRE

    Mayorov, Alexander V.; Cai, Minying; Palmer, Erin S.; Tanaka, Dustin K.; Cain, James P.; Dedek, Matthew M.; Tan, Bahar; Trivedi, Dev; Victor J. Hruby

    2011-01-01

    A novel hybrid melanocortin pharmacophore was designed based on the topographical similarities between the pharmacophores of Agouti related protein (AGRP) an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. When employed in two different 23-membered macrocyclic lactam peptide templates, the designed hybrid AGRP/MSH pharmacophore yielded non-competitive ligands with nanomolar range binding affinities. The topography-based pha...

  4. Kinetics of binding of dihydropyridine calcium channel ligands to skeletal muscle membranes: Evidence for low-affinity sites and for the involvement of G proteins

    International Nuclear Information System (INIS)

    Detailed kinetic studies of the binding of the calcium channel antagonist (+)-[3H]PN200-110 to membrane preparations form rabbit skeletal muscle have demonstrated that, in addition to the high-affinity sites that are readily measured in equilibrium and kinetic experiments, there are also dihydropyridine binding sites with much lower affinities. These sites were detected by the ability of micromolar concentrations of several dihydropyridines to accelerate the rate of dissociation of (+)-[3H]PN200-110 from its high-affinity sites. The observed increase in rate was dependent on the concentration of competing ligand, and half-maximal effects occurred at approximately 10 μM for the agonist (±)-Bay K8644 and for the antagonists nifedipine, (±)-nitrendipine, and (+)-PN200-110. The low-affinity sites appear to be stereospecific since (-)-PN200-110 (1-200 μM) did not affect the dissociation rate. The possible involvement of guanine nucleotide binding proteins in dihydropyridine binding has been investigated by studying the effects of guanosine 5'-O-(3-thiotriphosphate) (GTPγS) and guanosine 5'-O-(2-thiodiphosphate) (GDPβS) on binding parameters. GTPγS did increase the ability of (±)-[3H]PN200-110. These results suggest that skeletal muscle dihydropyridine receptors have low-affinity binding sites that may be involved in the regulation of calcium channel function and that activation of a guanine nucleotide binding protein may modulate the binding of agonists but not of antagonists to these sites

  5. A simple enzyme-substrate localized conjugation method to generate immobilized, functional glutathione S-transferase fusion protein columns for affinity enrichment.

    Science.gov (United States)

    Coughlin, John; Masci, Allyson; Gronke, Robert S; Bergelson, Svetlana; Co, Carl

    2016-07-15

    Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme-substrate site-specific cross-linking reaction; GSH-Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)-GST. The immobilized FcγRIIIa-GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity. PMID:27063248

  6. Analysis of drug-protein binding using on-line immunoextraction and high-performance affinity microcolumns: Studies with normal and glycated human serum albumin.

    Science.gov (United States)

    Matsuda, Ryan; Jobe, Donald; Beyersdorf, Jared; Hage, David S

    2015-10-16

    A method combining on-line immunoextraction microcolumns with high-performance affinity chromatography (HPAC) was developed and tested for use in examining drug-protein interactions with normal or modified proteins. Normal human serum albumin (HSA) and glycated HSA were used as model proteins for this work. High-performance immunoextraction microcolumns with sizes of 1.0-2.0 cm × 2.1mm i.d. and containing anti-HSA polyclonal antibodies were developed and tested for their ability to bind normal HSA or glycated HSA. These microcolumns were able to extract up to 82-93% for either type of protein at 0.05-0.10 mL/min and had a binding capacity of 0.34-0.42 nmol HSA for a 1.0 cm × 2.1mm i.d. microcolumn. The immunoextraction microcolumns and their adsorbed proteins were tested for use in various approaches for drug binding studies. Frontal analysis was used with the adsorbed HSA/glycated HSA to measure the overall affinities of these proteins for the drugs warfarin and gliclazide, giving comparable values to those obtained previously using similar protein preparations that had been covalently immobilized within HPAC columns. Zonal elution competition studies with gliclazide were next performed to examine the specific interactions of this drug at Sudlow sites I and II of the adsorbed proteins. These results were also comparable to those noted in prior work with covalently immobilized samples of normal HSA or glycated HSA. These experiments indicated that drug-protein binding studies can be carried out by using on-line immunoextraction microcolumns with HPAC. The same method could be used in the future with clinical samples and other drugs or proteins of interest in pharmaceutical studies or biomedical research.

  7. Novel peptide sequence ("IQ-tag" with high affinity for NIR fluorochromes allows protein and cell specific labeling for in vivo imaging.

    Directory of Open Access Journals (Sweden)

    Kimberly A Kelly

    Full Text Available BACKGROUND: Probes that allow site-specific protein labeling have become critical tools for visualizing biological processes. METHODS: Here we used phage display to identify a novel peptide sequence with nanomolar affinity for near infrared (NIR (benzindolium fluorochromes. The developed peptide sequence ("IQ-tag" allows detection of NIR dyes in a wide range of assays including ELISA, flow cytometry, high throughput screens, microscopy, and optical in vivo imaging. SIGNIFICANCE: The described method is expected to have broad utility in numerous applications, namely site-specific protein imaging, target identification, cell tracking, and drug development.

  8. A tandem laboratory scale protein purification process using Protein A affinity and anion exchange chromatography operated in a weak partitioning mode.

    Science.gov (United States)

    Shamashkin, Michael; Godavarti, Ranga; Iskra, Timothy; Coffman, Jon

    2013-10-01

    A significant consequence of scaling up production of high titer monoclonal antibody (mAb) processes in existing facilities is the generation of in-process pools that exceed the capacity of storage vessels. A semi-continuous downstream process where columns and filters are linked and operated in tandem would eliminate the need for intermediate holding tanks. This study is a bench-scale demonstration of the feasibility of a tandem process for the purification of mAbs employing an affinity Protein A capture step, followed by a flow-through anion-exchange (AEX) step with the possibility of adding an in-line virus filtration step (VF). All three steps were linked sequentially and operated as one continuous process using an ÄKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX column was operated in a weak partitioning (WP) mode enabled by a precise in-line titration of Protein A effluent. In order to avoid complex control schemes and facilitate validation, quality and robustness were built into the system through selection of buffers based on thermodynamic and empirical models. The tandem system utilized the simplest possible combination of valves, pumps, controls, and automation, so that it could easily be implemented in a clinical or commercial production facility. Linking the purification steps in a tandem process is expected to generate savings in time and production costs and also reduce the size of quality systems due to reduced documentation requirements, microbial sampling, and elimination of hold time validation. PMID:23633385

  9. Integrative refolding and purification of histidine-tagged protein by like-charge facilitated refolding and metal-chelate affinity adsorption.

    Science.gov (United States)

    Liu, Hu; Du, Wen-Jie; Dong, Xiao-Yan; Sun, Yan

    2014-05-30

    This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.

  10. Affinity Purification of Insulin by Peptide-Ligand Affinity Chromatography

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The affinity heptapeptide (HWWWPAS) for insulin, selected from phage display library,was coupled to EAH Sepharose 4B gel and packed to a 1-mL column. The column was used for the affinity purification of insulin from protein mixture and commercial insulin preparation. It was observed that the minor impurity in the commercial insulin was removed by the affinity chromatography. Nearly 40 mg of insulin could be purified with the 1-mL affinity column. The results revealed the high specificity and capacity of the affinity column for insulin purification. Moreover, based on the analysis of the amino acids in the peptide sequence, shorter peptides were designed and synthesized for insulin chromatography. As a result, HWWPS was found to be a good alternative to HWWWPAS, while the other two peptides with three or four amino acids showed weak affinity for insulin. The results indicated that the peptide sequence of HWWWPAS was quite conservative for specific binding of insulin.

  11. Affinity chromatography—dependent selection (ACDS) of genomic DNA fragments bound specifically to bacterial synthesized Myc/Myn proteins

    Institute of Scientific and Technical Information of China (English)

    SHICAN; PEIWANG; 等

    1995-01-01

    This paper describes an approach to seek for mouse c-Myc/Myn proteins-bound specific sequences among genomic DNA.cDNA fragment of myn gene was obtained through RT-PCR technique from RNA of NIH3T3 cells.DNA fragments encoding BR/HLH/LZ structure of Myc and Myn proteins were cloned in frame into pGEX-2T vector respectively.Fusion GST-Myc and GST-Myn synthesized in E.coli hosts showed affinity to CACGTG E-box DNA and subsequently interacted with genomic fragments prepared through whole-genome-PCR.A PCR-assisted procedure which combines protein-DNA interaction and affinity chromatography was designed to enrich Myc/Myn bound DNA.At least two genomic DNA fragments obtained exhibit specifical binding capacity to Myc/Myn complex but not to GST alone.Significance of the work and of the technique itself as well asidentification of the DNAs are discussed.

  12. Monoclonal antibody affinity purification of a 78 kDa membrane protein of Leishmania donovani of Indian origin and its role in host–parasite interaction

    Indian Academy of Sciences (India)

    Mandira Mukherjee; Anindita Bhattacharyya; Swadesh Duttagupta

    2002-12-01

    Monoclonal antibodies were raised against pathogenic promastigotes of Leishmania donovani of Indian origin. Among these, one was used for immuno-affinity purification of a 78 kDa membrane protein present in both the amastigote and promastigote forms of the parasite. Results of immunoblot experiments with the anti-78 kDa antibody revealed that the protein was present only in parasites belonging to the L. donovani complex. The expression of the protein was observed to be the same during different phases of growth of the promastigotes. Therefore, the 78 kDa protein is neither stage-specific nor differentially regulated. Surface iodination and subcellular fractionation of the promastigotes indicated that the protein was localized on the cell surface. The 78 kDa protein was found to inhibit the binding of promastigotes to macrophages significantly, suggesting that it may play a role in the process of infection. Thus, here we report the purification of a surface protein of L. donovani of Indian origin, which may play an important role in the process of infection.

  13. Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

    Science.gov (United States)

    Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

    2000-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

  14. Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity.

    Science.gov (United States)

    Czikora, Agnes; Lundberg, Daniel J; Abramovitz, Adelle; Lewin, Nancy E; Kedei, Noemi; Peach, Megan L; Zhou, Xiaoling; Merritt, Raymond C; Craft, Elizabeth A; Braun, Derek C; Blumberg, Peter M

    2016-05-20

    The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [(3)H]phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn(7), Ser(8), Ala(19), and Ile(21) with the corresponding residues from RasGRP1/3 (Thr(7), Tyr(8), Gly(19), and Leu(21), respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [(3)H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. PMID:27022025

  15. Affinity of ribosomal protein S8 from mesophilic and (hyper)thermophilic archaea and bacteria for 16S rRNA correlates with the growth temperatures of the organisms.

    Science.gov (United States)

    Gruber, Thomas; Köhrer, Caroline; Lung, Birgit; Shcherbakov, Dmitri; Piendl, Wolfgang

    2003-08-14

    The ribosomal protein S8 plays a pivotal role in the assembly of the 30S ribosomal subunit. Using filter binding assays, S8 proteins from mesophilic, and (hyper)thermophilic species of the archaeal genus Methanococcus and from the bacteria Escherichia coli and Thermus thermophilus were tested for their affinity to their specific 16S rRNA target site. S8 proteins from hyperthermophiles exhibit a 100-fold and S8 from thermophiles exhibit a 10-fold higher affinity than their mesophilic counterparts. Thus, there is a striking correlation of affinity of S8 proteins for their specific RNA binding site and the optimal growth temperatures of the respective organisms. The stability of individual rRNA-protein complexes might modulate the stability of the ribosome, providing a maximum of thermostability and flexibility at the growth temperature of the organism.

  16. Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads for magnetic affinity separation of histidine-tagged proteins.

    Science.gov (United States)

    Vereshchagina, T A; Fedorchak, M A; Sharonova, O M; Fomenko, E V; Shishkina, N N; Zhizhaev, A M; Kudryavtsev, A N; Frank, L A; Anshits, A G

    2016-01-28

    Magnetic Ni(2+)-zeolite/ferrosphere and Ni(2+)-silica/ferrosphere beads (Ni-ferrosphere beads - NFB) of a core-shell structure were synthesized starting from coal fly ash ferrospheres having diameters in the range of 0.063-0.050 mm. The strategy of NFB fabrication is an oriented chemical modification of the outer surface preserving the magnetic core of parent beads with the formation of micro-mesoporous coverings. Two routes of ferrosphere modification were realized, such as (i) hydrothermal treatment in an alkaline medium resulting in a NaP zeolite layer and (ii) synthesis of micro-mesoporous silica on the glass surface using conventional methods. Immobilization of Ni(2+) ions in the siliceous porous shell of the magnetic beads was carried out via (i) the ion exchange of Na(+) for Ni(2+) in the zeolite layer or (ii) deposition of NiO clusters in the zeolite and silica pores. The final NFB were tested for affinity in magnetic separation of the histidine-tagged green fluorescent protein (GFP) directly from a cell lysate. Results pointed to the high affinity of the magnetic beads towards the protein in the presence of 10 mM EDTA. The sorption capacity of the ferrosphere-based Ni-beads with respect to GFP was in the range 1.5-5.7 mg cm(-3).

  17. Affinity Capillary Electrophoresis:Study of the Binding of HIV-1 gp41 with a Membrane Protein (P45) on the Human B Cell Line,Raji

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Affinity capillary electrophoresis has been used to study the interaction between a membrane protein (P45) isolated from the Human B cell line, Raji, and rsgp41. P45, rsgp41 and the complexes were well resolved. The entire separation was achieved in less than 3min. Formations of two kinds of stable P45-rsgp41 complexes were confirmed based on migration time comparison; the binding equilibrium was achieved as soon as two proteins were mixed. The results indicate that the interaction between P45 and rsgp41 is strong with a fast association rate and a slow dissociation rate, and there are at least two kinds of binding sites with different binding constants between P45 and rsgp41.

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

    Science.gov (United States)

    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.

  19. Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation.

    Science.gov (United States)

    Burdman, S; Dulguerova, G; Okon, Y; Jurkevitch, E

    2001-04-01

    The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

  20. Assessment of GM-CSF receptors by real-time RT-PCR on cell lines expressing high and low affinity receptors and their relation to cytotoxic effect of chimeric protein (StxA1-GM-CSF

    Directory of Open Access Journals (Sweden)

    Habibi Roudkenar M.

    2007-06-01

    Full Text Available Immunotoxins, which are composed of both the cell targeting and the cell killing moieties are the new approach for targeted therapy of human disease .In all immunotoxins that GM-CSF has been used as cell targeting; only cell lines expressing high affinity receptor have been used for cytotoxicity studies. In the present study, various cell lines expressing high and low affinity receptors were used for assessment of the cytotoxic effect of hybrid chimeric protein. The expression of GM-CSF receptor (GM-CSFR was quantified by real-time RT- PCR. The cell lines K562 and THP1 expressing high affinity receptor and MC-7, PC-3 and DU145 expressing low affinity receptor were used for this study. The chimeric hybrid protein was found to be toxic for various cell lines used in this investigation and cytotoxicity was more effective in cell lines bearing high affinity receptors. Overall, our results showed that the recombinant hybrid protein could have wide range of application on various cancer cell lines even cells bearing low affinity receptors for GM-CSF.

  1. Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry

    DEFF Research Database (Denmark)

    Poulsen, Jon Wriedt; Madsen, Christian Toft; Young, Clifford;

    2013-01-01

    Protein digestion is an integral part of the "shotgun" proteomics approach and commonly requires overnight incubation prior to mass spectrometry analysis. Quadruplicate "shotgun" proteomic analysis of whole yeast lysate demonstrated that Guanidine-Hydrochloride (Gnd-HCl) protein digestion can...... be optimally completed within 30 min with endoprotease Lys-C. No chemical artifacts were introduced when samples were incubated in Gnd-HCl at 95 °C, making Gnd-HCl an appropriate digestion buffer for shotgun proteomics. Current methodologies for investigating protein-protein interactions (PPIs) often require....... To validate the Gnd-HCl approach, label-free PPI analysis of several GFP-tagged yeast deubiquitinating enzymes was performed. The identification of known interaction partners demonstrates the utility of the optimized Gnd-HCl protocol that is also scalable to the 96 well-plate format....

  2. Binding affinity between dietary polyphenols and β-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed.

    Science.gov (United States)

    Stojadinovic, Marija; Radosavljevic, Jelena; Ognjenovic, Jana; Vesic, Jelena; Prodic, Ivana; Stanic-Vucinic, Dragana; Cirkovic Velickovic, Tanja

    2013-02-15

    Non-covalent interactions between β-lactoglobulin (BLG) and polyphenol extracts of teas, coffee and cocoa were studied by fluorescence and CD spectroscopy at pH values of the gastrointestinal tract (GIT). The biological implications of non-covalent binding of polyphenols to BLG were investigated by in vitro pepsin and pancreatin digestibility assay and ABTS radical scavenging activity of complexes formed. The polyphenol-BLG systems were stable at pH values of the GIT. The most profound effect of pH on binding affinity was observed for polyphenol extracts rich in phenolic acids. Stronger non-covalent interactions delayed pepsin and pancreatin digestion of BLG and induced β-sheet to α-helix transition at neutral pH. All polyphenols tested protected protein secondary structure at an extremely acidic pH of 1.2. A positive correlation was found between the strength of protein-polyphenol interactions and (a) half time of protein decay in gastric conditions (R(2)=0.85), (b) masking of total antioxidant capacity of protein-polyphenol complexes (R(2)=0.95).

  3. mCSM-lig: quantifying the effects of mutations on protein-small molecule affinity in genetic disease and emergence of drug resistance.

    Science.gov (United States)

    Pires, Douglas E V; Blundell, Tom L; Ascher, David B

    2016-01-01

    The ability to predict how a mutation affects ligand binding is an essential step in understanding, anticipating and improving the design of new treatments for drug resistance, and in understanding genetic diseases. Here we present mCSM-lig, a structure-guided computational approach for quantifying the effects of single-point missense mutations on affinities of small molecules for proteins. mCSM-lig uses graph-based signatures to represent the wild-type environment of mutations, and small-molecule chemical features and changes in protein stability as evidence to train a predictive model using a representative set of protein-ligand complexes from the Platinum database. We show our method provides a very good correlation with experimental data (up to ρ = 0.67) and is effective in predicting a range of chemotherapeutic, antiviral and antibiotic resistance mutations, providing useful insights for genotypic screening and to guide drug development. mCSM-lig also provides insights into understanding Mendelian disease mutations and as a tool for guiding protein design. mCSM-lig is freely available as a web server at http://structure.bioc.cam.ac.uk/mcsm_lig. PMID:27384129

  4. [Prognosis of affinity change of the TATA-binding protein to TATA-boxes upon polymorphisms of the human gene promoter TATA boxes].

    Science.gov (United States)

    Ponomarenko, P M; Ponomarenko, M P; Drachkova, I A; Lysova, M V; Arshinova, T V; Savinkova, L K; Kolchanov, N A

    2009-01-01

    TATA-binding protein (TBP) is a subunit of basal transcription factor TFIID that recognizes and binds to the TATA-box on TATA-containing promoters of class II genes, and starts assembling RNA polymerase II basal transcription complex. It is shown in many works that the sequence of TATA-box with its flanking regions affects the level of basal and activated transcription. TATA-box polymorphisms and human hereditary diseases associated with them show that TBP/TATA interaction may indirectly affect gene regulation in vivo. The object of this work is to determine changes in the TBP/TATA affinity upon polymorphisms in TATA-boxes of human gene promoters. We assess changes in TBP/TATA affinities in silico by using our formula of equilibrium TBP/TATA binding upon four consecutive steps: nonspecific binding sliding braking (stopping) stabilization. Our prognoses agree with known examples of TATA-box polymorphisms and human hereditary diseases associated with them. PMID:19548537

  5. Protein–Protein interaction site prediction in Homo sapiens and E. coli using an interaction-affinity based membership function in fuzzy SVM

    Indian Academy of Sciences (India)

    Brijesh Kumar Sriwastava; Subhadip Basu; Ujjwal Maulik

    2015-10-01

    Protein–protein interaction (PPI) site prediction aids to ascertain the interface residues that participate in interaction processes. Fuzzy support vector machine (F-SVM) is proposed as an effective method to solve this problem, and we have shown that the performance of the classical SVM can be enhanced with the help of an interaction-affinity based fuzzy membership function. The performances of both SVM and F-SVM on the PPI databases of the Homo sapiens and E. coli organisms are evaluated and estimated the statistical significance of the developed method over classical SVM and other fuzzy membership-based SVM methods available in the literature. Our membership function uses the residue-level interaction affinity scores for each pair of positive and negative sequence fragments. The average AUC scores in the 10-fold cross-validation experiments are measured as 79.94% and 80.48% for the Homo sapiens and E. coli organisms respectively. On the independent test datasets, AUC scores are obtained as 76.59% and 80.17% respectively for the two organisms. In almost all cases, the developed F-SVM method improves the performances obtained by the corresponding classical SVM and the other classifiers, available in the literature.

  6. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  7. Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

    Science.gov (United States)

    Trisciuoglio, D; Desideri, M; Farini, V; De Luca, T; Di Martile, M; Tupone, M G; Urbani, A; D'Aguanno, S; Del Bufalo, D

    2016-01-01

    Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1-4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem-loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding. PMID:26866271

  8. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    Science.gov (United States)

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves. PMID:27129432

  9. Grafting iminodiacetic acid on silica nanoparticles for facilitated refolding of like-charged protein and its metal-chelate affinity purification.

    Science.gov (United States)

    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.

  10. A C1q domain containing protein from Crassostrea gigas serves as pattern recognition receptor and opsonin with high binding affinity to LPS.

    Science.gov (United States)

    Jiang, Shuai; Li, Hui; Zhang, Daoxiang; Zhang, Huan; Wang, Lingling; Sun, Jinsheng; Song, Linsheng

    2015-08-01

    C1q proteins serve as pattern recognition receptors and involve in the pathogen recognition and complement pathway activation. In the present study, a novel C1q domain containing protein from Crassostrea gigas (designated CgC1qDC-1) was isolated by liposaccharide-Sepharose 6B affinity chromatography. The coding sequence of CgC1qDC-1 gene was determined by performing a homologous search of eight tryptic peptides identified by MALDI-TOF/TOF-MS against the genome of C. gigas. The coding sequence of CgC1qDC-1 was of 387 bp encoding a polypeptide of 128 amino acids containing a typical globular C1q domain. The globular C1q domain possessed eight β strands with a jelly-roll topology structure, which was similar to the structure of human gC1q domain. The mRNA transcripts of CgC1qDC-1 were dominantly expressed in mantle and hemocytes, while low expressed in hepatopancreas, gonad, gill and muscle. The expression level of CgC1qDC-1 increased drastically at 6 h after Vibrio splendidus stimulation, and then gradually fell to the normal level at about 24 h. ELISA assay quantified that CgC1qDC-1 bound to LPS with high binding affinity (Kd = 0.09 × 10(-6) M). Moreover, CgC1qDC-1 significantly enhanced the phagocytosis of oyster hemocytes towards Gram-negative bacteria Escherichia coli and V. splendidus. These results collectively indicated that CgC1qDC-1 could serve as pattern recognition receptor and opsonin in the innate immune response against invading Gram-negative bacteria.

  11. Tag Selection of Tandem Affinity Purification and Its Application in Plant Protein-Protein Interactions%TAP亲和标签选择及其在植物蛋白互作研究中的应用

    Institute of Scientific and Technical Information of China (English)

    张志飞; 赵志丽; 文昭竹

    2014-01-01

    Tandem affinity purification(TAP)is a technology for the purification of protein complex under the normal physiological conditions, which has been extensively utilized to identify protein-protein interactions(PPIs)and reveal interaction network of protein complexes. In recent years, TAP technology has been improving in the PPIs study, with the development of new labels, TAP method improvements and jointed use with other technologies associated. TAP technology is also being increasingly used in the studies of plant PPIs. This paper reviews the selection of TAP affinity tag and advance of research that TAP has been successful used in the study of plant PPIs.%串联亲和纯化法(TAP)是一种纯化生理条件下蛋白复合物的技术,已广泛应用于鉴定蛋白相互作用和揭示蛋白复合物互作网络。近年来,随着TAP新型标签的不断出现以及与其他技术的联用,TAP技术正逐渐应用于植物蛋白质互作研究中。综述了TAP亲和标签选择,并介绍其在植物蛋白互作研究中的成功应用。

  12. High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

    Science.gov (United States)

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  13. An electronic environment and contact direction sensitive scoring function for predicting affinities of protein-ligand complexes in Contour(®).

    Science.gov (United States)

    Lindblom, Peter R; Wu, Guosheng; Liu, Zhijie; Jim, Kam-Chuen; Baldwin, John J; Gregg, Richard E; Claremon, David A; Singh, Suresh B

    2014-09-01

    Contour(®) is a computational structure-based drug design technology that grows drug-like molecules by assembling context sensitive fragments in well-defined binding pockets. The grown molecules are scored by a novel empirical scoring function developed using high-resolution crystal structures of diverse classes of protein-ligand complexes and associated experimental binding affinities. An atomic model bearing features of the valence bond and VSEPR theories embodying their molecular electronic environment has been developed for non-covalent intermolecular interactions. On the basis of atomic hybridization and polarization states, each atom is modeled by features representing electron lone pairs, p-orbitals, and polar and non-polar hydrogens. A simple formal charge model was used to differentiate between polar and non-polar atoms. The interaction energy and the desolvation contribution of the protein-ligand association energy is computed as a linear sum of pair-wise interactions and desolvation terms. The pair-wise interaction energy captures short-range positive electrostatic interactions via hydrogen bonds, electrostatic repulsion of like charges, and non-bond contacts. The desolvation energy is estimated by calculating the energy required to desolvate interaction surfaces of the protein and the ligand in the complex. The scoring function predicts binding energies of a diverse set of protein-ligand complexes used for training with a correlation coefficient of 0.61. It also performs equally well in predicting association energies of a diverse validation set of protein-ligand complexes with a correlation coefficient of 0.57, which is equivalent to or better than 12 other scoring functions tested against this set including X-Score, GOLD, and DrugScore. PMID:25123650

  14. The purification of affinity-labelled active-site peptides

    International Nuclear Information System (INIS)

    The isolation of the labelled peptide from the protein digest, following the affinity labelling of the active sites of enzymes or antibodies, is described. Single-step affinity chromatography utilises the affinity of the native enzymes or antibody for the ligand used to label the same protein. The labelled peptide is the only one in the digest that displays affinity for the immobilised protein and can be released with eluants that dissociate the protein-ligand complex. (Auth.)

  15. Expression of Major Capsid Protein of Cainine Parvovirus by Yeast (Pichia pastoris and Efficient Purification using Arginine in Affinity Chromatography

    Directory of Open Access Journals (Sweden)

    Ying He

    2016-06-01

    Full Text Available An immunochromatographic (IC assay was developed for rapid detection of canine parvovirus using the monoclonal antibodies (McAbs against canine parvovirus (CPV-2. To prepare the McAbs, gene encoding the VP2 protein of CPV-2a was expressed in a Pichia pastoris expression vector pPICZ-A. The recombinant VP2 was similar antigenically function to the native capsid protein as demonstrated by Western blotting using CPV- 2 polyclonal antiserum. McAbs against CPV-2 were produced by fusing myeloma cell line SP2/0 with spleen cells from Balb/C mice immunized with purified recombinant VP2 protein. By ELISA it was shown that the McAbs specifically recognized VP2 epitopes of CPV-2 but not those of other canine viruses such as Canine distemper virus (CDV or canine adenovirus (CAV. An IC assay developed with the McAbs was suitable for rapid detection of canine parvovirus. Fecal samples (120 from dogs suspected of CPV-2 infection were analyzed by both haemaglutination (HA assay and the IC assay, and 52 and 53 samples were found positive for CPV-2, respectively. Comparison between the two different assays revealed that IC assay is as sensitive as HA; the sensitivity and specificity for the IC assay is 98.6% and 98.1%, respectively.

  16. Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

    Science.gov (United States)

    Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M.; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

    2016-01-01

    The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

  17. Proteína bruta e energia digestível em dietas para alevinos de curimbatá (Prochilodus affins Crude protein and digestible energy in the diets for curimbatá fingerlings (Prochilodus affins

    Directory of Open Access Journals (Sweden)

    Marcos Antonio Delmondes Bomfim

    2005-12-01

    Full Text Available Objetivando-se determinar as exigências de proteína bruta (PB em função do nível de energia digestível (ED da dieta para alevinos de curimbatá (Prochilodus affins, foram utilizados 256 peixes com peso inicial de 2,72 ± 0,11 g, mantidos em 32 aquários de 100 L, dotados de abastecimento de água, temperatura controlada e aeração individuais. O experimento foi realizado seguindo-se esquema fatorial 4 x 2 (quatro níveis de PB: 18,0; 22,0; 26,0; e 30,0%, combinados com dois níveis de ED: 2.700 e 3.000 kcal/kg, em um delineamento inteiramente ao acaso, com quatro repetições e oito peixes por unidade experimental. Os peixes foram alimentados ad libitum em duas refeições diárias durante 78 dias. Avaliaram-se o ganho de peso, o consumo aparente de racão, a conversão alimentar aparente, a taxa de crescimento específico, a taxa de eficiência protéica, a eficiência de retenção de nitrogênio, a umidade, a proteína e a gordura corporais e as porcentagens de nitrogênio e de gordura no ganho de peso. Verificou-se que não houve interação dos níveis de PB ´ ED, à exceção da porcentagem de gordura no ganho de peso. Com o nível de 3.000 kcal de ED/kg, obtiveram-se peixes com maiores níveis de matéria seca, gordura corporal e porcentagem de gordura no ganho de peso. Concluiu-se que a exigência de PB e ED para alevinos de curimbatá é de 26,05% e 2.700 kcal/kg, respectivamente, que corresponde a uma relação ED:PB de 10,36 kcal de ED/g de PB, por proporcionar as melhores respostas em ganho de peso e composição de carcaça.Two hundred and fifty six curimbatá (Prochilodus affins fingerlings averaging initial weight of 2.72 ± 0.11 g, stocked in 32 aquariums (100 L with water renewal, controlled temperature and individual aeration, were used to determine the dietary crude protein (CP requirements, according to the digestible energy level (DE. The experiment was carried out in to a 4 x 2 factorial scheme (four CP levels: 18

  18. Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae) through Affinity Chromatography.

    Science.gov (United States)

    Feng, Mingxing; He, Zhenyu; Wang, Yuanyuan; Yan, Xiufang; Zhang, Jiwen; Hu, Zhaonong; Wu, Wenjun

    2016-01-01

    Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH) sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH) dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2) oxygenase superfamily protein) were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS) analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests. PMID:27153092

  19. The roles of the hybrid cluster protein, Hcp and its reductase, Hcr, in high affinity nitric oxide reduction that protects anaerobic cultures of Escherichia coli against nitrosative stress.

    Science.gov (United States)

    Wang, Jing; Vine, Claire E; Balasiny, Basema K; Rizk, John; Bradley, Charlene L; Tinajero-Trejo, Mariana; Poole, Robert K; Bergaust, Linda L; Bakken, Lars R; Cole, Jeffrey A

    2016-06-01

    The hybrid cluster protein, Hcp, contains a 4Fe-2S-2O iron-sulfur-oxygen cluster that is currently considered to be unique in biology. It protects various bacteria from nitrosative stress, but the mechanism is unknown. We demonstrate that the Escherichia coli Hcp is a high affinity nitric oxide (NO) reductase that is the major enzyme for reducing NO stoichiometrically to N2 O under physiologically relevant conditions. Deletion of hcp results in extreme sensitivity to NO during anaerobic growth and inactivation of the iron-sulfur proteins, aconitase and fumarase, by accumulated cytoplasmic NO. Site directed mutagenesis revealed an essential role in NO reduction for the conserved glutamate 492 that coordinates the hybrid cluster. The second gene of the hcp-hcr operon encodes an NADH-dependent reductase, Hcr. Tight interaction between Hcp and Hcr was demonstrated. Although Hcp and Hcr purified individually were inactive or when recombined, a co-purified preparation reduced NO in vitro with a Km for NO of 500 nM. In an hcr mutant, Hcp is reversibly inactivated by NO concentrations above 200 nM, indicating that Hcr protects Hcp from nitrosylation by its substrate, NO. PMID:26879449

  20. Candidate SNP Markers of Chronopathologies Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

    Directory of Open Access Journals (Sweden)

    Petr Ponomarenko

    2016-01-01

    Full Text Available Variations in human genome (e.g., single nucleotide polymorphisms, SNPs may be associated with hereditary diseases, their complications, comorbidities, and drug responses. Using Web service SNP_TATA_Comparator presented in our previous paper, here we analyzed immediate surroundings of known SNP markers of diseases and identified several candidate SNP markers that can significantly change the affinity of TATA-binding protein for human gene promoters, with circadian consequences. For example, rs572527200 may be related to asthma, where symptoms are circadian (worse at night, and rs367732974 may be associated with heart attacks that are characterized by a circadian preference (early morning. By the same method, we analyzed the 90 bp proximal promoter region of each protein-coding transcript of each human gene of the circadian clock core. This analysis yielded 53 candidate SNP markers, such as rs181985043 (susceptibility to acute Q fever in male patients, rs192518038 (higher risk of a heart attack in patients with diabetes, and rs374778785 (emphysema and lung cancer in smokers. If they are properly validated according to clinical standards, these candidate SNP markers may turn out to be useful for physicians (to select optimal treatment for each patient and for the general population (to choose a lifestyle preventing possible circadian complications of diseases.

  1. Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA

    International Nuclear Information System (INIS)

    Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes. (author)

  2. Candidate SNP Markers of Chronopathologies Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

    Science.gov (United States)

    Ponomarenko, Petr; Rasskazov, Dmitry; Suslov, Valentin; Sharypova, Ekaterina; Savinkova, Ludmila; Podkolodnaya, Olga; Podkolodny, Nikolay L.; Tverdokhleb, Natalya N.; Chadaeva, Irina; Kolchanov, Nikolay

    2016-01-01

    Variations in human genome (e.g., single nucleotide polymorphisms, SNPs) may be associated with hereditary diseases, their complications, comorbidities, and drug responses. Using Web service SNP_TATA_Comparator presented in our previous paper, here we analyzed immediate surroundings of known SNP markers of diseases and identified several candidate SNP markers that can significantly change the affinity of TATA-binding protein for human gene promoters, with circadian consequences. For example, rs572527200 may be related to asthma, where symptoms are circadian (worse at night), and rs367732974 may be associated with heart attacks that are characterized by a circadian preference (early morning). By the same method, we analyzed the 90 bp proximal promoter region of each protein-coding transcript of each human gene of the circadian clock core. This analysis yielded 53 candidate SNP markers, such as rs181985043 (susceptibility to acute Q fever in male patients), rs192518038 (higher risk of a heart attack in patients with diabetes), and rs374778785 (emphysema and lung cancer in smokers). If they are properly validated according to clinical standards, these candidate SNP markers may turn out to be useful for physicians (to select optimal treatment for each patient) and for the general population (to choose a lifestyle preventing possible circadian complications of diseases).

  3. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    Science.gov (United States)

    Bardwell, A J; Flatauer, L J; Matsukuma, K; Thorner, J; Bardwell, L

    2001-03-30

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo. PMID:11134045

  4. Influence of the high-affinity growth hormone (GH)-binding protein on plasma profiles of free and bound GH and on the apparent half-life of GH. Modeling analysis and clinical applications.

    OpenAIRE

    Veldhuis, J D; Johnson, M.L.; Faunt, L M; MERCADO, M.; Baumann, G

    1993-01-01

    The discovery of a specific high-affinity growth hormone (GH) binding protein (GH-BP) in plasma adds complexity to the dynamics of GH secretion and clearance. Intuitive predictions are that such a protein would damp sharp oscillations in GH concentrations otherwise caused by bursts of GH secretion into the blood volume, prolong the apparent half-life of circulating GH, and contribute a reservoir function. To test these implicit considerations, we formulated an explicit mathematical model of p...

  5. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

    Science.gov (United States)

    Bigenzahn, Johannes W; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K; Scorzoni, Stefania; Vladimer, Gregory I; Müller, André C; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L; Superti-Furga, Giulio

    2016-03-01

    Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.

  6. Nuclear DNA sensor IFI16 as circulating protein in autoimmune diseases is a signal of damage that impairs endothelial cells through high-affinity membrane binding.

    Directory of Open Access Journals (Sweden)

    Francesca Gugliesi

    Full Text Available IFI16, a nuclear pathogenic DNA sensor induced by several pro-inflammatory cytokines, is a multifaceted protein with various functions. It is also a target for autoantibodies as specific antibodies have been demonstrated in the sera of patients affected by systemic autoimmune diseases. Following transfection of virus-derived DNA, or treatment with UVB, IFI16 delocalizes from the nucleus to the cytoplasm and is then eventually released into the extracellular milieu. In this study, using an in-house capture enzyme-linked immunsorbent assay we demonstrate that significant levels of IFI16 protein can also exist as circulating form in the sera of autoimmune patients. We also show that the rIFI16 protein, when added in-vitro to endothelial cells, does not affect cell viability, but severely limits their tubulogenesis and transwell migration activities. These inhibitory effects are fully reversed in the presence of anti-IFI16 N-terminal antibodies, indicating that its extracellular activity resides within the N-terminus. It was further demonstrated that endogenous IFI16 released by apoptotic cells bind neighboring cells in a co-culture. Immunofluorescence assays revealed existence of high-affinity binding sites on the plasma membrane of endothelial cells. Free recombinant IFI16 binds these sites on HUVEC with dissociation constant of 2.7 nM, radioiodinated and unlabeled IFI16 compete for binding sites, with inhibition constant (Ki of 14.43 nM and half maximal inhibitory concentration (IC50 of 67.88 nM; these data allow us to estimate the presence of 250,000 to 450,000 specific binding sites per cell. Corroborating the results from functional assays, this binding could be completely inhibited using anti-IFI16 N-terminal antibody, but not with an antibody raised against the IFI16 C-terminal. Altogether, these data demonstrate that IFI16 may exist as circulating protein in the sera of autoimmune patients which binds endothelial cells causing damage

  7. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    Science.gov (United States)

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β.

  8. Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin Prevent the Expression of Proinflammatory Chemokines and Cytokines in Pigs

    Directory of Open Access Journals (Sweden)

    R. Frandoloso

    2013-01-01

    Full Text Available The expression of chemokines (CCL-2 and CXCL-8 and cytokines (IL-1α, IL-1β, IL-6, TNF-α, and IL-10 was evaluated by RT-qPCR in colostrum-deprived pigs vaccinated and challenged with Haemophilus parasuis serovar 5. Two vaccines containing native proteins with affinity to porcine transferrin (NPAPTim and NPAPTit were tested, along with two control groups: one inoculated with PBS instead of antigen (challenge group (CHG, and another one nonimmunized and noninfected (blank group. The use of NPAPTim and NPAPTit resulted in complete protection against H. parasuis (no clinical signs and/or lesions, and both vaccines were capable of avoiding the expression of the proinflammatory molecules to levels similar to physiological values in blank group. However, overexpression of all proinflammatory molecules was observed in CHG group, mainly in the target infection tissues (brain, lungs, and spleen. High expression of CCL-2, CXCL-8, IL-1α, IL-1β, and IL-6 can be considered one of the characteristics of H. parasuis infection by serovar 5.

  9. The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity

    DEFF Research Database (Denmark)

    Hansen, B E; Nielsen, Claus Henrik; Madsen, H O;

    2011-01-01

    Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype...

  10. Synthesis and Evaluation of In Vitro DNA/Protein Binding Affinity, Antimicrobial, Antioxidant and Antitumor Activity of Mononuclear Ru(II) Mixed Polypyridyl Complexes.

    Science.gov (United States)

    Putta, Venkat Reddy; Chintakuntla, Nagamani; Mallepally, Rajender Reddy; Avudoddi, Srishailam; K, Nagasuryaprasad; Nancherla, Deepika; V V N, Yaswanth; R S, Prakasham; Surya, Satyanarayana Singh; Sirasani, Satyanarayana

    2016-01-01

    The four novel Ru(II) complexes [Ru(phen)2MAFIP](2+) (1) [MAFIP = 2-(5-(methylacetate)furan-2-yl)-1 H-imidazo[4,5-f] [1, 10]phenanthroline, phen = 1,10-Phenanthroline], [Ru(bpy)2MAFIP](2+) (2) (bpy = 2,2'-bipyridine) and [Ru(dmb)2MAFIP](2+) (3) (dmb = 4,4'-dimethyl-2,2'-bipyridine) and [Ru(hdpa)2MAFIP](2+) (4) (hdpa = 2,2-dipyridylamine) have been synthesized and fully characterized via elemental analysis, NMR spectroscopy, EI-MS and FT-IR spectroscopy. In addition, the DNA-binding behaviors of the complexes 1-4 with calf thymus DNA were investigated by UV-Vis absorption, fluorescence studies and viscosity measurement. The DNA-binding experiments showed that the complexes 1-4 interact with CT-DNA through an intercalative mode. BSA protein binding affinity of synthesized complexes was determined by UV/Vis absorption and fluorescence emission titrations. The binding affinity of ruthenium complexes was supported by molecular docking. The photoactivated cleavage of plasmid pBR322 DNA by ruthenium complexes 1-4 was investigated. All the synthesized compounds were tested for antimicrobial activity by using three Gram-negative (Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa) and three Gram-positive (Micrococcus luteus, Bacillus subtilis and Bacillus megaterium) organisms, these results indicated that complex 3 was more activity compared to other complexes against all tested microbial strains while moderate antimicrobial activity profile was noticed for complex 4. The antioxidant activity experiments show that the complexes exhibit moderate antioxidant activity. The cytotoxicity of synthesized complexes on HeLa cell lines has been examined by MTT assay. The apoptosis assay was carried out with Acridine Orange (AO) staining methods and the results indicate that complexes can induce the apoptosis of HeLa cells. The cell cycle arrest investigated by flow cytometry and these results indicate that complexes 1-4 induce the cell cycle arrest at G0/G1

  11. High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

    Science.gov (United States)

    Prasad, Joni M; Young, Patricia A; Strickland, Dudley K

    2016-08-26

    The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor. PMID:27402839

  12. Protein isolation using affinity chromatography

    NARCIS (Netherlands)

    Besselink, T.

    2012-01-01

    Many product or even waste streams in the food industry contain components that may have potential for e.g. functional foods. These streams are typically large in volume and the components of interest are only present at low concentrations. A robust and highly selective separation process should be

  13. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    Science.gov (United States)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-09-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  14. High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein-Protein Interaction

    Energy Technology Data Exchange (ETDEWEB)

    He, Shihan; Senter, Timothy J.; Pollock, Jonathan; Han, Changho; Upadhyay, Sunil Kumar; Purohit, Trupta; Gogliotti, Rocco D.; Lindsley, Craig W.; Cierpicki, Tomasz; Stauffer, Shaun R.; Grembecka, Jolanta [Michigan; (Vanderbilt); (Vanderbilt-MED)

    2014-10-02

    The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ~288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

  15. Focal cerebral ischaemia induces a decrease in activity and a shift in ouabain affinity of Na+, K+-ATPase isoforms without modifications in mRNA and protein expression.

    Science.gov (United States)

    Jamme, I; Barbey, O; Trouvé, P; Charlemagne, D; Maixent, J M; MacKenzie, E T; Pellerin, L; Nouvelot, A

    1999-02-20

    In a mouse model of focal cerebral ischaemia, we observed after 1 h of ischaemia, that the total Na+, K+-ATPase activity was decreased by 39.4%, and then did not vary significantly up to 6 h post-occlusion. In the sham group, the dose-response curves for ouabain disclosed three inhibitory sites of low (LA), high (HA) and very high (VHA) affinity. In ischaemic animals, we detected the presence of only two inhibitory sites for ouabain. After 1 h of permanent occlusion, the first site exhibited a low affinity while the second site presented an affinity intermediate between those of HA and VHA sites, which evolved after 3 h and 6 h of occlusion towards that of the VHA site. The presence of only two ouabain sites for Na+, K+-ATPase after ischaemia could result from a change in ouabain affinity of both HA and VHA sites (alpha2 and alpha3 isoforms, respectively) to form a unique component. Irrespective of the duration of ischaemia, the smaller activity of this second site accounted entirely for the loss in total activity. Surprisingly, no modifications in protein and mRNA expression of any alpha or beta isoforms of the enzyme were observed, thus suggesting that ischaemia could induce intrinsic modifications of the Na+, K+-ATPase. PMID:10082868

  16. Affine Grassmann codes

    DEFF Research Database (Denmark)

    Høholdt, Tom; Beelen, Peter; Ghorpade, Sudhir Ramakant

    2010-01-01

    We consider a new class of linear codes, called affine Grassmann codes. These can be viewed as a variant of generalized Reed-Muller codes and are closely related to Grassmann codes.We determine the length, dimension, and the minimum distance of any affine Grassmann code. Moreover, we show that...... affine Grassmann codes have a large automorphism group and determine the number of minimum weight codewords....

  17. Removing Endotoxin from Protein Solution by Chitosan Modified Affinity Membrane%壳聚糖亲和膜脱除蛋白质溶液中内毒素

    Institute of Scientific and Technical Information of China (English)

    孙海翔; 张林; 柴红; 陈欢林

    2005-01-01

    Affinity membrane was prepared with chitosan immobilized on the hydrophile- modified poly(vinylidene fluoride) (PVDF) membrane. Fourier transform infrared spectroscopy (FTIR) analysis indicated that the contents of-NH2 and -OH groups increased and fluoride decreased on the membrane surface after modification. Using this kind of affinity membrane, the effects of operation parameters such as pH, ionic strength and flow rate, on the amount of endotoxin removed were investigated. The results showed that the equilibrium adsorption capacity and the dissociation constant of the affinity membrane to endotoxin were 21.4 EU·mg-1 membrane and 0.50 EU·ml-1,respectively, at pH 7.0 and ionic strength 0.2 mol·L-1. Adsorption appeared to follow a typical Langmuir adsorption isotherm. At pH 5.0, ionic strength of 0.2 mol·L-1, the removal rate of endotoxin from BSA solution with the chitosan affinity membrane was up to 88.6% (11.50 EU·mg-1 membrane), and the recovery of BSA was 93.4% (0.187 mg·mg-1 membrane), while at pH 11.0, ionic strength of 0.2 mol·L-1, the removal rate of endotoxin from lysozyme solution was 72.4% (9.92 EU·mg- 1 membrane), and the recovery of lysozyme was 92.3% (0.104 mg·mg- 1 membrane).

  18. [Analysis of the impact of heparin on the affinity of high mobility group box-1 protein and the receptor of advanced glycation end products by surface plasmon resonance technology].

    Science.gov (United States)

    Ling, Yan; Wang, Chun-You; Yang, Zhi-Yong

    2009-11-01

    To investigate the affinity constants of heparin with high mobility group protein 1(HMGB1) and HMGB1 with the receptor of advanced glycation end products (RAGE) and to analyze the impact of heparin on the affinity of HMGB1 and RAGE, the standard BIAcore amine coupling chemistry protocol using EDC and NHS was employed for immobilizing. Surface plasmon resonance biosensor technology was used to detect the affinity constants of heparin/HMGB1, HMGB1/RAGE and heparin/ RAGE. Binding analysis was used to investigate the impact of heparin on the affinity of HMGB1 and RAGE. After the immobilization, 9 000 and 5 000 RU rise of HMGB1 and RAGE respectively were obtained. These meant that the immobilized values of HMGB1 and RAGE were about 9 and 5 ng x mm(-2) respectively. The kinetic constants were k(a) = 1.78 x 10(5) L x mol(-1) x s(-1), kd = 8.02 x 10(-4) s(-1), and the affinity constants were KA = 2.22 x 10(8) L x mol(-1), the equilibrium dissociation constant K(D) = 4.5 x 10(-9) mol x L(-1) for heparin and HMGB1; while the kinetic constants were k(a) = 1.85 x 10(3) L x mol(-1) x s(-1), k(d) = 1.81 x 10(-4) s(-1), K(A) = 1.02 x 10(7) L x mol(-1), K(D) = 9.77 x 10(-8) mol x L(-1) for HMGB1 and RAGE; there was very low affinity of heparin with RAGE. The highest concentration of 10 000 u x L(-1) of heparin in this experiment did not reach the saturation with HMGB1. After 50 mg x L(-1) of HMGB1 was mixed with heparin of 50, 100, 1 000, 10 000 u x L(-1), the combining amount of HMGB1 and RAGE declined from 100 to 50 RU. But there were no significant differences between different concentrations of heparin. It was concluded that heparin can combine with HMGB1 and affect the affinity of HMGB1/RAGE. In addition, this impact was not in a dose-dependent manner. PMID:20101991

  19. The connection between metal ion affinity and ligand affinity in integrin I domains

    DEFF Research Database (Denmark)

    Vorup-Jensen, Thomas; Waldron, TT; Astrof, N;

    2007-01-01

    Integrins are cell-surface heterodimeric proteins that mediate cell-cell, cell-matrix, and cell-pathogen interactions. Half of the known integrin alpha subunits contain inserted domains (I domains) that coordinate ligand through a metal ion. Although the importance of conformational changes within...... isolated I domains in regulating ligand binding has been reported, the relationship between metal ion binding affinity and ligand binding affinity has not been elucidated. Metal and ligand binding by several I domain mutants that are stabilized in different conformations are investigated using isothermal...... titration calorimetry and surface plasmon resonance studies. This work suggests an inverse relationship between metal ion affinity and ligand binding affinity (i.e. constructs with a high affinity for ligand exhibit a low affinity for metal). This trend is discussed in the context of structural studies...

  20. European and international collaboration in affinity proteomics.

    Science.gov (United States)

    Stoevesandt, Oda; Taussig, Michael J

    2012-06-15

    In affinity proteomics, specific protein-binding molecules (a.k.a. binders), principally antibodies, are applied as reagents in proteome analysis. In recent years, advances in binder technologies have created the potential for an unprecedented view on protein expression and distribution patterns in plasma, cells and tissues and increasingly on protein function. Particular strengths of affinity proteomics methods include detecting proteins in their natural environments of cell or tissue, high sensitivity and selectivity for detection of low abundance proteins and exploiting binding actions such as functional interference in living cells. To maximise the use and impact of affinity reagents, it will be essential to create comprehensive, standardised binder collections. With this in mind, the EU FP7 programme AFFINOMICS (http://www.affinomics.org), together with the preceding EU programmes ProteomeBinders and AffinityProteome, aims to extend affinity proteomics research by generating a large-scale resource of validated protein-binding molecules for characterisation of the human proteome. Activity is directed at producing binders to about 1000 protein targets, primarily in signal transduction and cancer, by establishing a high throughput, coordinated production pipeline. An important aspect of AFFINOMICS is the development of highly efficient recombinant selection methods, based on phage, cell and ribosome display, capable of producing high quality binders at greater throughput and lower cost than hitherto. The programme also involves development of innovative and sensitive technologies for specific detection of target proteins and their interactions, and deployment of binders in proteomics studies of clinical relevance. The need for such binder generation programmes is now recognised internationally, with parallel initiatives in the USA for cancer (NCI) and transcription factors (NIH) and within the Human Proteome Organisation (HUPO). The papers in this volume of New

  1. 软骨藻酸作用蛋白质的亲和毛细管电泳筛选%Affinity capillary electrophoresis for screening proteins interacting with domoic acid

    Institute of Scientific and Technical Information of China (English)

    王晓倩; 高铁; 洪专; 屈锋

    2015-01-01

    基于亲和毛细管电泳,定性比较了血浆、肠道及细胞线粒体中所选的9种重要功能蛋白质与神经毒性生物毒素———软骨藻酸( domoic acid,DA)的相互作用。以 DA淌度比变化量ΔM 与蛋白质浓度 L 作图,根据斜率值大小可比较 DA与蛋白质作用的相对强弱。结果如下:可与 DA相互作用的有6种蛋白质,作用强弱为:人凝血酶>细胞色素 C≈胰蛋白酶≈免疫球蛋白 E( Ig E)≈核糖核酸酶 A>λ核酸外切酶;而铁蛋白、转铁蛋白、凝集素与 DA未表现出相互作用和亲和力。实验表明,亲和毛细管电泳具有高效、快速、所需样品量低的优点,可用于DA作用靶蛋白的筛选,为 DA的毒性机制研究和毒性防护提供基础信息。%Domoic acid( DA)is a biological neurotoxin that causes amnesic shellfish poison-ing. Study of the interactions between DA and important functional proteins contributes to understand the toxicity mechanism of DA to biological macromolecules. In this paper,the inter-actions between DA and nine important proteins in plasma,intestine and mitochondria were qualitatively compared by affinity capillary electrophoresis. Proteins were used as affinity lig-ands while DA as the affinity receptor. Proteins with the concentrations of 0,0. 2,0. 4,0. 6, 0. 8 μmol/L were added in the electrophoresis buffer and the migration times of 0. 2 mg/mL DA were detected. Then the linear graphs of the variation of DA mobility ratio(ΔM)with the pro-tein mass concentration( L)were drawn. According to the slope value,the relative strength of the interactions between DA and proteins was compared. The results showed that six proteins can interact with DA and the relative strength order was human thrombin>cytochrome C≈tryp-sin≈immunoglobulin E( Ig E)≈ribonuclease A>λ exonuclease,while ferritin,transferrin and lectin had no affinity with DA. With the advantages of high efficiency,fast analysis and less

  2. Affine dynamics with torsion

    Energy Technology Data Exchange (ETDEWEB)

    Gueltekin, Kemal [Izmir Institute of Technology, Department of Physics, Izmir (Turkey)

    2016-03-15

    In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schroedinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor. (orig.)

  3. A novel recombinant AzrC protein proposed by molecular docking and in silico analyses to improve azo dye's binding affinity.

    Science.gov (United States)

    Dehghanian, Fariba; Kay, Maryam; Kahrizi, Danial

    2015-09-15

    Azo dyes are broadly used in different industries through their chemical stability and ease of synthesis. These dyes are usually identified as critical environmental pollutants and many attentions were performed to degradation of azo dyes using biological systems. In this study, the interactions of an azoreductase from mesophilic gram-positive Bacillus sp. B29, AzrC, with four common azo dyes (orange I, orange II, orange G and acid red 88) were investigated. Fifteen points, double, triple and quadruple mutant forms of AzrC were made using Molegro Virtual Docker 6.0 in order to improve the binding affinity of azo dyes to AzrC. The impact of 15 different mutations on azo dye affinity potency of AzrC was computationally analyzed using AzrC-azo dye molecular docking, and each interaction was scored based on AutoDock 4.2 free binding energy. Our results have indicated that Asn 104 (A), Asn 187 (B), and Tyr 151 (A) make stable hydrogen bond between AzrC and azo dyes. The hydrophobic amino acids like Phe105 (A), Phe 125 (B), and Phe 172 (B) in wild type form make hydrophobic interactions. In addition, the presence of more hydrophobic residues F60 (B), I119 (B), I121 (B) and F132 (B) in mutant forms made more powerful hydrophobic pocket in the active site. In conclusion, recombinant AzrC with quadruple mutations was suggested in order to increase the biodegradation capacity of AzrC through improving its affinity to four studied azo dyes. This study would be promising for future experimental analyses in order to produce recombinant form of AzrC.

  4. A novel recombinant AzrC protein proposed by molecular docking and in silico analyses to improve azo dye's binding affinity.

    Science.gov (United States)

    Dehghanian, Fariba; Kay, Maryam; Kahrizi, Danial

    2015-09-15

    Azo dyes are broadly used in different industries through their chemical stability and ease of synthesis. These dyes are usually identified as critical environmental pollutants and many attentions were performed to degradation of azo dyes using biological systems. In this study, the interactions of an azoreductase from mesophilic gram-positive Bacillus sp. B29, AzrC, with four common azo dyes (orange I, orange II, orange G and acid red 88) were investigated. Fifteen points, double, triple and quadruple mutant forms of AzrC were made using Molegro Virtual Docker 6.0 in order to improve the binding affinity of azo dyes to AzrC. The impact of 15 different mutations on azo dye affinity potency of AzrC was computationally analyzed using AzrC-azo dye molecular docking, and each interaction was scored based on AutoDock 4.2 free binding energy. Our results have indicated that Asn 104 (A), Asn 187 (B), and Tyr 151 (A) make stable hydrogen bond between AzrC and azo dyes. The hydrophobic amino acids like Phe105 (A), Phe 125 (B), and Phe 172 (B) in wild type form make hydrophobic interactions. In addition, the presence of more hydrophobic residues F60 (B), I119 (B), I121 (B) and F132 (B) in mutant forms made more powerful hydrophobic pocket in the active site. In conclusion, recombinant AzrC with quadruple mutations was suggested in order to increase the biodegradation capacity of AzrC through improving its affinity to four studied azo dyes. This study would be promising for future experimental analyses in order to produce recombinant form of AzrC. PMID:26026905

  5. Lectin affinity chromatography of glycolipids

    Energy Technology Data Exchange (ETDEWEB)

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  6. Affine and degenerate affine BMW algebras: Actions on tensor space

    CERN Document Server

    Daugherty, Zajj; Virk, Rahbar

    2012-01-01

    The affine and degenerate affine Birman-Murakami-Wenzl (BMW) algebras arise naturally in the context of Schur-Weyl duality for orthogonal and symplectic quantum groups and Lie algebras, respectively. Cyclotomic BMW algebras, affine and cyclotomic Hecke algebras, and their degenerate versions are quotients. In this paper we explain how the affine and degenerate affine BMW algebras are tantalizers (tensor power centralizer algebras) by defining actions of the affine braid group and the degenerate affine braid algebra on tensor space and showing that, in important cases, these actions induce actions of the affine and degenerate affine BMW algebras. We then exploit the connection to quantum groups and Lie algebras to determine universal parameters for the affine and degenerate affine BMW algebras. Finally, we show that the universal parameters are central elements--the higher Casimir elements for orthogonal and symplectic enveloping algebras and quantum groups.

  7. A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F

    2005-08-25

    Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid

  8. Short communication: Identification of iron-binding peptides from whey protein hydrolysates using iron (III)-immobilized metal ion affinity chromatography and reversed phase-HPLC-tandem mass spectrometry.

    Science.gov (United States)

    Cruz-Huerta, Elvia; Martínez Maqueda, Daniel; de la Hoz, Lucia; da Silva, Vera S Nunes; Pacheco, Maria Teresa Bertoldo; Amigo, Lourdes; Recio, Isidra

    2016-01-01

    Peptides with iron-binding capacity obtained by hydrolysis of whey protein with Alcalase (Novozymes, Araucaria, PR, Brazil), pancreatin, and Flavourzyme (Novozymes) were identified. Hydrolysates were subjected to iron (III)-immobilized metal ion affinity chromatography, and the bound peptides were sequenced by mass spectrometry. Regardless of the enzyme used, the domains f(42-59) and f(125-137) from β-lactoglobulin enclosed most of identified peptides. This trend was less pronounced in the case of peptides derived from α-lactalbumin, with sequences deriving from diverse regions. Iron-bound peptides exhibited common structural characteristics, such as an abundance of Asp, Glu, and Pro, as revealed by mass spectrometry and AA analysis. In conclusion, this characterization of iron-binding peptides helps clarify the relationship between peptide structure and iron-chelating activity and supports the promising role of whey protein hydrolysates as functional ingredients in iron supplementation treatments. PMID:26601589

  9. Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

    Directory of Open Access Journals (Sweden)

    Mladkova Jana

    2010-08-01

    Full Text Available Abstract Background Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs and other proteins interacting with glutathione (GSH in model cell lines could be of particular interest. Methods We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE. We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. Results Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. Conclusions Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

  10. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  11. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins

    DEFF Research Database (Denmark)

    Bruun, A W; Svendsen, I; Sørensen, S O;

    1998-01-01

    degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several...

  12. Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size

    DEFF Research Database (Denmark)

    Knudsen, J; Faergeman, N J; Skøtt, H;

    1994-01-01

    Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitut...... resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis....

  13. Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

    OpenAIRE

    Bieberich, Erhard

    2011-01-01

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins bindin...

  14. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    OpenAIRE

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.; Landry, Samuel J.

    2014-01-01

    Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-te...

  15. Congophilicity (Congo red affinity) of different beta2-microglobulin conformations characterized by dye affinity capillary electrophoresis

    DEFF Research Database (Denmark)

    Heegaard, N H; Sen, J W; Nissen, Mogens Holst

    2000-01-01

    The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red-affinities of......The amyloidogenic protein beta-microglobulin was characterized by affinity capillary electrophoresis (CE). CE could separate conformational variants of beta2-microglobulin and with the amyloid-specific dye Congo red as a buffer additive it was possible to measure different Congo red......-affinities of native and abnormally folded beta2-microglobulin. We find that native beta2-microglobulin has an intermediate affinity for Congo red at pH 7.3 and that binding involves electrostatic interactions. The conformational variant of beta2-microglobulin that appears in acetonitrile solutions binds Congo red...... more strongly. Affinity CE using Congo red as a buffer additive is a new, simple, fast, and quantitative micromethod for the characterization of soluble conformational intermediates of amyloidogenic proteins....

  16. Affine stochastic mortality

    NARCIS (Netherlands)

    D.F. Schrager

    2006-01-01

    We propose a new model for stochastic mortality. The model is based on the literature on affine term structure models. It satisfies three important requirements for application in practice: analytical tractibility, clear interpretation of the factors and compatibility with financial option pricing m

  17. Affinity purification of aprotinin from bovine lung.

    Science.gov (United States)

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  18. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

    Directory of Open Access Journals (Sweden)

    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  19. Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

    Directory of Open Access Journals (Sweden)

    Hunt Kelly K

    2006-03-01

    Full Text Available Abstract Background Isotope-coded affinity tag (ICAT tandem mass spectrometry (MS allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers was also performed. Results 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42, beta-globin (H:L ratio 1.98, hemopexin (H:L ratio 1.73, and vitamin D-binding protein precursor (H:L ratio 1.82 were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

  20. Affine and degenerate affine BMW algebras: The center

    CERN Document Server

    Daugherty, Zajj; Virk, Rahbar

    2011-01-01

    The degenerate affine and affine BMW algebras arise naturally in the context of Schur-Weyl duality for orthogonal and symplectic Lie algebras and quantum groups, respectively. Cyclotomic BMW algebras, affine Hecke algebras, cyclotomic Hecke algebras, and their degenerate versions are quotients. In this paper the theory is unified by treating the orthogonal and symplectic cases simultaneously; we make an exact parallel between the degenerate affine and affine cases via a new algebra which takes the role of the affine braid group for the degenerate setting. A main result of this paper is an identification of the centers of the affine and degenerate affine BMW algebras in terms of rings of symmetric functions which satisfy a "cancellation property" or "wheel condition" (in the degenerate case, a reformulation of a result of Nazarov). Miraculously, these same rings also arise in Schubert calculus, as the cohomology and K-theory of isotropic Grassmanians and symplectic loop Grassmanians. We also establish new inte...

  1. ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

    International Nuclear Information System (INIS)

    Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with K m of 56.36 ± 0.27 μM and V max of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with K i of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism

  2. Capillary electrophoresis-based assessment of nanobody affinity and purity

    NARCIS (Netherlands)

    Haselberg, Rob; Oliveira, Sabrina; van der Meel, Roy; Somsen, Govert W; de Jong, Gerhardus J

    2014-01-01

    Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a heavy-chain-

  3. Hierarchical Affinity Propagation

    CERN Document Server

    Givoni, Inmar; Frey, Brendan J

    2012-01-01

    Affinity propagation is an exemplar-based clustering algorithm that finds a set of data-points that best exemplify the data, and associates each datapoint with one exemplar. We extend affinity propagation in a principled way to solve the hierarchical clustering problem, which arises in a variety of domains including biology, sensor networks and decision making in operational research. We derive an inference algorithm that operates by propagating information up and down the hierarchy, and is efficient despite the high-order potentials required for the graphical model formulation. We demonstrate that our method outperforms greedy techniques that cluster one layer at a time. We show that on an artificial dataset designed to mimic the HIV-strain mutation dynamics, our method outperforms related methods. For real HIV sequences, where the ground truth is not available, we show our method achieves better results, in terms of the underlying objective function, and show the results correspond meaningfully to geographi...

  4. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    linker for yeast surface display of scFv and scFab fragments, we compared a series of different Gly-Ser-based linkers in display and antigen binding proficiency. We show that these formats of the model antibody can accommodate linkers of different lengths and that introduction of alanine or glutamate......-2. Based on the presented data we suggest that affinity maturation of the model antibody proceeds through multiple incremental steps of subtle improvements. We moreover conclude that the best affinity improved candidates are likely to be obtained through optimization of both the antigen...... fragments by in vivo homologous recombination large combinatorial antibody libraries can easily be generated. We have optimized ordered assembly of three CDR fragments into a gapped vector and observed increased transformation efficiency in a yeast strain carrying a deletion of the SGS1 helicase...

  5. Gaussian Affine Feature Detector

    OpenAIRE

    Xu, Xiaopeng; Zhang, Xiaochun

    2011-01-01

    A new method is proposed to get image features' geometric information. Using Gaussian as an input signal, a theoretical optimal solution to calculate feature's affine shape is proposed. Based on analytic result of a feature model, the method is different from conventional iterative approaches. From the model, feature's parameters such as position, orientation, background luminance, contrast, area and aspect ratio can be extracted. Tested with synthesized and benchmark data, the method achieve...

  6. Affinity driven social networks

    Science.gov (United States)

    Ruyú, B.; Kuperman, M. N.

    2007-04-01

    In this work we present a model for evolving networks, where the driven force is related to the social affinity between individuals of a population. In the model, a set of individuals initially arranged on a regular ordered network and thus linked with their closest neighbors are allowed to rearrange their connections according to a dynamics closely related to that of the stable marriage problem. We show that the behavior of some topological properties of the resulting networks follows a non trivial pattern.

  7. Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster.

    OpenAIRE

    Pellerin, I.; Schnabel, C; Catron, K M; Abate, C

    1994-01-01

    The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five re...

  8. Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I.

    OpenAIRE

    Jones, J. I.; D'Ercole, A J; Camacho-Hubner, C; Clemmons, D R

    1991-01-01

    The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human...

  9. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    Science.gov (United States)

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  10. Comprehensive Analysis of Contributions from Protein Conformational Stability and Major Histocompatibility Complex Class II-Peptide Binding Affinity to CD4+ Epitope Immunogenicity in HIV-1 Envelope Glycoprotein

    Science.gov (United States)

    Li, Tingfeng; Steede, N. Kalaya; Nguyen, Hong-Nam P.; Freytag, Lucy C.; McLachlan, James B.; Mettu, Ramgopal R.; Robinson, James E.

    2014-01-01

    ABSTRACT Helper T-cell epitope dominance in human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is not adequately explained by peptide binding to major histocompatibility complex (MHC) proteins. Antigen processing potentially influences epitope dominance, but few, if any, studies have attempted to reconcile the influences of antigen processing and MHC protein binding for all helper T-cell epitopes of an antigen. Epitopes of gp120 identified in both humans and mice occur on the C-terminal flanks of flexible segments that are likely to be proteolytic cleavage sites. In this study, the influence of gp120 conformation on the dominance pattern in gp120 from HIV strain 89.6 was examined in CBA mice, whose MHC class II protein has one of the most well defined peptide-binding preferences. Only one of six dominant epitopes contained the most conserved element of the I-Ak binding motif, an aspartic acid. Destabilization of the gp120 conformation by deletion of single disulfide bonds preferentially enhanced responses to the cryptic I-Ak motif-containing sequences, as reported by T-cell proliferation or cytokine secretion. Conversely, inclusion of CpG in the adjuvant with gp120 enhanced responses to the dominant CD4+ T-cell epitopes. The gp120 destabilization affected secretion of some cytokines more than others, suggesting that antigen conformation could modulate T-cell functions through mechanisms of antigen processing. IMPORTANCE CD4+ helper T cells play an essential role in protection against HIV and other pathogens. Thus, the sites of helper T-cell recognition, the dominant epitopes, are targets for vaccine design; and the corresponding T cells may provide markers for monitoring infection and immunity. However, T-cell epitopes are difficult to identify and predict. It is also unclear whether CD4+ T cells specific for one epitope are more protective than T cells specific for other epitopes. This work shows that the three-dimensional (3D) structure of an

  11. Affine morphisms at zero level

    CERN Document Server

    Das, Paramita; Gupta, Ved Prakash

    2010-01-01

    Given a finite index subfactor, we show that the {\\em affine morphisms at zero level} in the affine category over the planar algebra associated to the subfactor is isomorphic to the fusion algebra of the subfactor as a *-algebra.

  12. On the Affine Isoperimetric Inequalities

    Indian Academy of Sciences (India)

    Wuyang Yu; Gangsong Leng

    2011-11-01

    We obtain an isoperimetric inequality which estimate the affine invariant -surface area measure on convex bodies. We also establish the reverse version of -Petty projection inequality and an affine isoperimetric inequality of $_{-p}K$.

  13. Adjoint affine fusion and tadpoles

    OpenAIRE

    Urichuk, Andrew; Walton, Mark A.

    2016-01-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-pol...

  14. Affine Patches on Positroid Varieties and Affine Pipe Dreams (Thesis)

    CERN Document Server

    Snider, Michelle

    2010-01-01

    The objects of interest in this thesis are positroid varieties in the Grassmannian, which are indexed by juggling patterns. In particular, we study affine patches on these positroid varieties. Our main result corresponds these affine patches to Kazhdan-Lusztig varieties in the affine Grassmannian. We develop a new term order and study how these spaces are related to subword complexes and Stanley-Reisner ideals. We define an extension of pipe dreams to the affine case and conclude by showing how our affine pipe dreams are generalizations of Cauchon and Le diagrams.

  15. Affine and quasi-affine frames for rational dilations

    DEFF Research Database (Denmark)

    Bownik, Marcin; Lemvig, Jakob

    2011-01-01

    , the corresponding family of quasi-affine systems are frames with uniform frame bounds. We also prove a similar equivalence result between pairs of dual affine frames and dual quasi-affine frames. Finally, we uncover some fundamental differences between the integer and rational settings by exhibiting an example......In this paper we extend the investigation of quasi-affine systems, which were originally introduced by Ron and Shen [J. Funct. Anal. 148 (1997), 408-447] for integer, expansive dilations, to the class of rational, expansive dilations. We show that an affine system is a frame if, and only if...

  16. Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors

    Institute of Scientific and Technical Information of China (English)

    Paul C. SEHNKE; Beth J. LAUGHNER; Carla R. LYERLY LINEBARGER; William B. GURLEY; Robert J.FERL

    2005-01-01

    Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs).The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood.In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A.thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

  17. Is It Reliable to Use Common Molecular Docking Methods for Comparing the Binding Affinities of Enantiomer Pairs for Their Protein Target?

    Science.gov (United States)

    Ramírez, David; Caballero, Julio

    2016-01-01

    Molecular docking is a computational chemistry method which has become essential for the rational drug design process. In this context, it has had great impact as a successful tool for the study of ligand-receptor interaction modes, and for the exploration of large chemical datasets through virtual screening experiments. Despite their unquestionable merits, docking methods are not reliable for predicting binding energies due to the simple scoring functions they use. However, comparisons between two or three complexes using the predicted binding energies as a criterion are commonly found in the literature. In the present work we tested how wise is it to trust the docking energies when two complexes between a target protein and enantiomer pairs are compared. For this purpose, a ligand library composed by 141 enantiomeric pairs was used, including compounds with biological activities reported against seven protein targets. Docking results using the software Glide (considering extra precision (XP), standard precision (SP), and high-throughput virtual screening (HTVS) modes) and AutoDock Vina were compared with the reported biological activities using a classification scheme. Our test failed for all modes and targets, demonstrating that an accurate prediction when binding energies of enantiomers are compared using docking may be due to chance. We also compared pairs of compounds with different molecular weights and found the same results. PMID:27104528

  18. PAS-1, a protein affinity purified from Ascaris suum worms, maintains the ability to modulate the immune response to a bystander antigen.

    Science.gov (United States)

    Oshiro, Telma M; Enobe, Cristina S; Araújo, Cláudia A; Macedo, Mahasti S; Macedo-Soares, Maria Fernanda

    2006-04-01

    Helminth infections and parasite components have potent immunomodulatory effects on a host's immune system. In the present study, we investigated the effect of PAS-1, a protein component of Ascaris suum adult worms recognized by a monoclonal antibody (MAIP-1), on humoral and cell-mediated responses to a bystander antigen (ovalbumin [OVA]). MAIP-1 recognized only one of the three polypeptide chains of PAS-1, but neutralized the suppressive effect of the whole worm extract on OVA-specific antibody production. PAS-1 inhibited antibody production against a T-cell-dependent, but not a T-cell-independent, antigen in a dose-dependent way. IgM, IgG1, IgG2b, and also IgE and anaphylactic IgG1 levels were downregulated. In addition, PAS-1 inhibited OVA-specific delayed type hypersensitivity reactions in the footpad of mice, showing a potent immunosuppressive activity on both Th1 and Th2 responses that seems to be mediated by the induction of large amounts of IL-10 and IL-4. Indeed, PAS-1-specific spleen cells secreted sevenfold more IL-10 and threefold more IL-4 than OVA-specific cells in response to in vitro restimulation with the respective antigens. In conclusion, we showed that PAS-1, a single protein component from A. suum, maintains all its immunosuppressive properties. PMID:16519731

  19. Virtual Screening of Plant Volatile Compounds Reveals a High Affinity of Hylamorpha elegans (Coleoptera: Scarabaeidae) Odorant-Binding Proteins for Sesquiterpenes From Its Native Host.

    Science.gov (United States)

    González-González, Angélica; Palma-Millanao, Rubén; Yáñez, Osvaldo; Rojas, Maximiliano; Mutis, Ana; Venthur, Herbert; Quiroz, Andrés; Ramírez, Claudio C

    2016-01-01

    Hylamorpha elegans(Burmeister) is a native Chilean scarab beetle considered to be a relevant agricultural pest to pasture and cereal and small fruit crops. Because of their cryptic habits, control with conventional methods is difficult; therefore, alternative and environmentally friendly control strategies are highly desirable. The study of proteins that participate in the recognition of odorants, such as odorant-binding proteins (OBPs), offers interesting opportunities to identify new compounds with the potential to modify pest behavior and computational screening of compounds, which is commonly used in drug discovery, may help to accelerate the discovery of new semiochemicals. Here, we report the discovery of four OBPs inH. elegans as well as six new volatiles released by its native host Nothofagus obliqua(Mirbel). Molecular docking performed between OBPs and new and previously reported volatiles from N. oblique revealed the best binding energy values for sesquiterpenic compounds. Despite remarkable divergence at the amino acid level, three of the four OBPs evaluated exhibited the best interaction energy for the same ligands. Molecular dynamics investigation reinforced the importance of sesquiterpenes, showing that hydrophobic residues of the OBPs interacted most frequently with the tested ligands, and binding free energy calculations demonstrated van der Waals and hydrophobic interactions to be the most important. Altogether, the results suggest that sesquiterpenes are interesting candidates for in vitro and in vivo assays to assess their potential application in pest management strategies.

  20. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    Science.gov (United States)

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association. PMID:26555266

  1. Expression of cold-adapted β-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

    Science.gov (United States)

    Kudou, Motonori; Okazaki, Fumiyoshi; Asai-Nakashima, Nanami; Ogino, Chiaki; Kondo, Akihiko

    2015-01-01

    Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes. PMID:25214227

  2. ZZ亲和肽-碱性磷酸酶融合蛋白的制备与初步应用%Preparation of fusion protein between ZZ affinity peptide and alkaline phosphatase and its primary application

    Institute of Scientific and Technical Information of China (English)

    鲍如梦; 赵爽佳; 薛敏; 杨洪鸣; 唐金宝

    2015-01-01

    目的:构建、表达ZZ亲和肽与碱性磷酸酶( AP)融合蛋白,并研究其生物学活性。方法:将ZZ亲和肽基因与AP基因重组,构建原核表达载体pEZZ-AP并利用大肠杆菌表达,金属离子亲和层析纯化目的蛋白,Western blot鉴定ZZ-AP生物学活性并初步应用于免疫细胞化学。结果:所构建重组质粒pEZZ-AP 经E.coli DH5α表达,SDS-PAGE结果显示目的蛋白相对分子量为62 kD,与理论值相近。 Western blot结果表明ZZ-AP融合蛋白既具有兔IgG抗体结合活性,又具有碱性磷酸酶活性,在细胞免疫组化应用中与AP标记的羊抗兔二抗显色模式与效果相似。结论:ZZ-AP融合蛋白构建成功,该融合蛋白具有兔IgG 抗体结合活性和碱性磷酸酶活性,细胞免疫组化应用结果表明该融合蛋白在免疫分析中具有潜在的应用价值。%Objective:To construct and express the fusion protein between ZZ affinity peptide and alkaline phosphatase and examine its biological activities.Methods:The alkaline phosphatase gene was cloned into pEZZ 18 vector containing ZZ peptide gene resulted in the pEZZ-AP recombinant vector.Then the vector was transformed and expressed in E.coli DH5α.And HisTrap affinity chromatography was employed to separate and purify the target protein.After analyzed by Western blot , ZZ-AP fusion protein was applied to immunocytochemistry as an alterative second antibody.Results:The result of SDS-PAGE showed that the fusion protein with a relative molecular weight was consistent with the theoretical values (62 kD).The fusion protein has rabbit IgG-binding ability and en-zymatic activity of alkaline phosphatase ,those were validated in Western blot;and it produced a good signal that was comparable in its staining pattern to that generated with goat anti-rabbit IgG-AP in immunocytochemistry.Conclusion: The ZZ-AP fusion protein was constructed successfully ,it has rabbit IgG-binding ability and enzymatic

  3. The Utility of Affine Variables and Affine Coherent States

    CERN Document Server

    Klauder, John R

    2011-01-01

    Affine coherent states are generated by affine kinematical variables much like canonical coherent states are generated by canonical kinematical variables. Although all classical and quantum formalisms normally entail canonical variables, it is shown that affine variables can serve equally well for many classical and quantum studies. This general purpose analysis provides tools to discuss two major applications: (1) the completely successful quantization of a nonrenormalizable scalar quantum field theory by affine techniques, in complete contrast to canonical techniques which only offer triviality; and (2) a formulation of the kinematical portion of quantum gravity that favors affine kinematical variables over canonical kinematical variables, and which generates a framework in which a favorable analysis of the constrained dynamical issues can take place. All this is possible because of the close connection between the affine and the canonical stories, while the few distinctions can be used to advantage when ap...

  4. Jacobi Structures on Affine Bundles

    Institute of Scientific and Technical Information of China (English)

    J. GRABOWSKI; D. IGLESIAS; J. C. MARRERO; E. PADR(O)N; P. URBA(N)SKI

    2007-01-01

    We study affine Jacobi structures (brackets) on an affine bundle π: A→M, i.e. Jacobi brackets that close on affine functions. We prove that if the rank of A is non-zero, there is a one-to- one correspondence between affine Jacobi structures on A and Lie algebroid structures on the vector bundle A+=∪p∈M Aff(Ap, R) of affine functionals. In the case rank A = 0, it is shown that there is a one-to-one correspondence between affins Jacobi structures on A and local Lie algebras on A+. Some examples and applications, also for the linear case, are discussed. For a special type of affine Jacobi structures which are canonically exhibited (strongly-affine or affine-homogeneous Jacobi structures) over a real vector space of finite dimension, we describe the leaves of its characteristic foliation as the orbits of an affine representation. These afline Jacobi structures can be viewed as an analog of the Kostant-Arnold-LiouviUe linear Poisson structure on the dual space of a real finite-dimensional Lie algebra.

  5. Fast preparation of antioxidant proteins from ginkgo and soybean by using affinity chromatography%亲和层析快速提取白果和大豆的抗氧化蛋白

    Institute of Scientific and Technical Information of China (English)

    王桂珍; 凌辉生; 谢丽思; 李任强

    2012-01-01

    After Sephadex G-25 polymerbeads were activated by using epichlorohydrin,hemin was bound with them to prepare an immobilized hemin affinity chromatography column,which was used to prepare antioxidant proteins from ginkgo and soybean.Equilibrated with deionized water and eluted with pH3.6,0.2mol/L NaAc-HAc buffer,those proteins obtained from this column were demonstrated to possess high antioxidative activity(AA) after the measurement with linoleic acid-potassium thiocyanate.AA of crude proteins of ginkgo was 7.92% and two polypeptides of them were obtained with AA 17.45% after purification.In soybean,several polypeptides with AA 30.72% were obtained from the crude proteins with AA 10.52%.This method was a novel,rapid and effective method for preparation of antioxidant proteins from ginkgo and soybean.%用环氧氯丙烷活化法把血红素结合于惰性分子筛填料SephadexG-25上作为配基做成亲和层析柱,用于从白果或大豆中提取具有抗氧化活性的蛋白。此层析柱以去离子水为平衡液、0.2mol/L的NaAc-HAc缓冲液(pH3.6)为洗脱液,层析得到的蛋白经亚油酸-硫氰酸钾抗氧化活性测定证明具有较高的抗氧化活性。白果全蛋白的抗氧化活性为7.92%,经此方法提纯后得到2种抗氧化多肽,活性达到17.45%;而大豆中的抗氧化蛋白经纯化则得到多种,抗氧化活性由全蛋白时的10.52%提高至30.72%。这是一个新的、只需一个步骤即可从白果或大豆蛋白提取液中提取到具有高抗氧化活性蛋白的方法,具有成本低,快速高效的特点。

  6. Adjoint affine fusion and tadpoles

    Science.gov (United States)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  7. Adjoint affine fusion and tadpoles

    CERN Document Server

    Urichuk, Andrew

    2016-01-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows, and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  8. Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2 with High Affinity Peptide Ligands Using Fluorescence Polarization

    Directory of Open Access Journals (Sweden)

    María Isabel Fernández-Bachiller

    2016-07-01

    Full Text Available Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

  9. How to Use SNP_TATA_Comparator to Find a Significant Change in Gene Expression Caused by the Regulatory SNP of This Gene’s Promoter via a Change in Affinity of the TATA-Binding Protein for This Promoter

    Directory of Open Access Journals (Sweden)

    Mikhail Ponomarenko

    2015-01-01

    Full Text Available The use of biomedical SNP markers of diseases can improve effectiveness of treatment. Genotyping of patients with subsequent searching for SNPs more frequent than in norm is the only commonly accepted method for identification of SNP markers within the framework of translational research. The bioinformatics applications aimed at millions of unannotated SNPs of the “1000 Genomes” can make this search for SNP markers more focused and less expensive. We used our Web service involving Fisher’s Z-score for candidate SNP markers to find a significant change in a gene’s expression. Here we analyzed the change caused by SNPs in the gene’s promoter via a change in affinity of the TATA-binding protein for this promoter. We provide examples and discuss how to use this bioinformatics application in the course of practical analysis of unannotated SNPs from the “1000 Genomes” project. Using known biomedical SNP markers, we identified 17 novel candidate SNP markers nearby: rs549858786 (rheumatoid arthritis; rs72661131 (cardiovascular events in rheumatoid arthritis; rs562962093 (stroke; rs563558831 (cyclophosphamide bioactivation; rs55878706 (malaria resistance, leukopenia, rs572527200 (asthma, systemic sclerosis, and psoriasis, rs371045754 (hemophilia B, rs587745372 (cardiovascular events; rs372329931, rs200209906, rs367732974, and rs549591993 (all four: cancer; rs17231520 and rs569033466 (both: atherosclerosis; rs63750953, rs281864525, and rs34166473 (all three: malaria resistance, thalassemia.

  10. Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.

    Science.gov (United States)

    Moua, Pachai S; Gonzalez, Alfonso; Oshiro, Kristin T; Tam, Vivian; Li, Zhiguo Harry; Chang, Jennifer; Leung, Wilson; Yon, Amy; Thor, Der; Venkatram, Sri; Franz, Andreas H; Risser, Douglas D; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2016-08-01

    The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.

  11. Realization of Fractal Affine Transformation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This paper gives the definition of fractal affine transformation and presents a specific method for its realization and its cor responding mathematical equations which are essential in fractal image construction.

  12. Representations of affine Hecke algebras

    CERN Document Server

    Xi, Nanhua

    1994-01-01

    Kazhdan and Lusztig classified the simple modules of an affine Hecke algebra Hq (q E C*) provided that q is not a root of 1 (Invent. Math. 1987). Ginzburg had some very interesting work on affine Hecke algebras. Combining these results simple Hq-modules can be classified provided that the order of q is not too small. These Lecture Notes of N. Xi show that the classification of simple Hq-modules is essentially different from general cases when q is a root of 1 of certain orders. In addition the based rings of affine Weyl groups are shown to be of interest in understanding irreducible representations of affine Hecke algebras. Basic knowledge of abstract algebra is enough to read one third of the book. Some knowledge of K-theory, algebraic group, and Kazhdan-Lusztig cell of Cexeter group is useful for the rest

  13. Infinite transitivity on affine varieties

    OpenAIRE

    Arzhantsev, Ivan; Flenner, Hubert; Kaliman, Shulim; Kutzschebauch, Frank; ZAIDENBERG, MIKHAIL

    2012-01-01

    In this note we survey recent results on automorphisms of affine algebraic varieties, infinitely transitive group actions and flexibility. We present related constructions and examples, and discuss geometric applications and open problems.

  14. The application of magnetic force differentiation for the measurement of the affinity of peptide libraries

    International Nuclear Information System (INIS)

    A new method has been developed for measuring the binding affinity of phage displayed peptides and a target protein using magnetic particles. The specific interaction between the phage displayed peptides and the target protein was subject to a force generated by the magnetic particle. The binding affinity was obtained by analyzing the force-bond lifetime

  15. The sodium ion affinities of asparagine, glutamine, histidine and arginine

    Science.gov (United States)

    Wang, Ping; Ohanessian, Gilles; Wesdemiotis, Chrys

    2008-01-01

    The sodium ion affinities of the amino acids Asn, Gln, His and Arg have been determined by experimental and computational approaches (for Asn, His and Arg). Na+-bound heterodimers with amino acid and peptide ligands (Pep1, Pep2) were produced by electrospray ionization. From the dissociation kinetics of these Pep1-Na+-Pep2 ions to Pep1-Na+ and Pep2-Na+, determined by collisionally activated dissociation, a ladder of relative affinities was constructed and subsequently converted to absolute affinities by anchoring the relative values to known Na+ affinities. The Na+ affinities of Asn, His and Arg, were calculated at the MP2(full)/6-311+G(2d,2p)//MP2/6-31G(d) level of ab initio theory. The resulting experimental and computed Na+ affinities are in excellent agreement with one another. These results, combined with those of our previous studies, yield the sodium ion affinities of 18 out of the 20 [alpha]-amino acids naturally occurring in peptides and proteins of living systems.

  16. Kinetic controlled affinity labeling of target enzyme with thioester chemistry.

    Science.gov (United States)

    Tomohiro, Takenori; Nakabayashi, Masahiro; Sugita, Yuka; Morimoto, Shota

    2016-08-01

    High specificity has been an important feature in affinity labeling for target profiling. Especially, to label targets via rapidly progressing reactions with consumption of ligand (probe), high specificity of reaction with common functional groups of target protein should be achieved without reactions with similar groups of non-target proteins. Herein, we demonstrate the kinetic controlled affinity labeling of acyl CoA synthetase using a fatty acid analogue containing a phenylthioester linkage. High specificity was attained by accelerating the labeling rate in the binding pocket. This approach could be useful for profiling a series of target enzymes and transporters in signal transduction pathways. PMID:27298000

  17. Several Affinity Tags Commonly Used in Chromatographic Purification

    Directory of Open Access Journals (Sweden)

    Xinyu Zhao

    2013-01-01

    Full Text Available Affinity tags have become powerful tools from basic biological research to structural and functional proteomics. They were widely used to facilitate the purification and detection of proteins of interest, as well as the separation of protein complexes. Here, we mainly discuss the benefits and drawbacks of several affinity or epitope tags frequently used, including hexahistidine tag, FLAG tag, Strep II tag, streptavidin-binding peptide (SBP tag, calmodulin-binding peptide (CBP, glutathione S-transferase (GST, maltose-binding protein (MBP, S-tag, HA tag, and c-Myc tag. In some cases, a large-size affinity tag, such as GST or MBP, can significantly impact on the structure and biological activity of the fusion partner protein. So it is usually necessary to excise the tag by protease. The most commonly used endopeptidases are enterokinase, factor Xa, thrombin, tobacco etch virus, and human rhinovirus 3C protease. The proteolysis features of these proteases are described in order to provide a general guidance on the proteolytic removal of the affinity tags.

  18. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    Science.gov (United States)

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%. PMID:26856529

  19. Protein-protein binding affinities calculated using the LIE method

    OpenAIRE

    Andberg, Tor Arne Heim

    2011-01-01

    Absolute binding free energies for the third domain of the turkey ovomucoid inhibitor in complex with Streptomyces griseus proteinase B and porcine pancreatic elastase has been calculated using the linear interaction energy method.

  20. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    Directory of Open Access Journals (Sweden)

    Renata Angeli

    2009-01-01

    Full Text Available A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A and Cratylia mollis (Cramoll 1 and Cramoll 1,4 did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column.

  1. Ferromagnetic levan composite: an affinity matrix to purify lectin.

    Science.gov (United States)

    Angeli, Renata; da Paz, Nathalia V N; Maciel, Jackeline C; Araújo, Flávia F B; Paiva, Patrícia M G; Calazans, Glícia M T; Valente, Ana Paula; Almeida, Fábio C L; Coelho, Luana C B B; Carvalho, Luiz B; Silva, Maria da Paz C; dos Santos Correia, Maria Tereza

    2009-01-01

    A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column. PMID:19547713

  2. Affine density in wavelet analysis

    CERN Document Server

    Kutyniok, Gitta

    2007-01-01

    In wavelet analysis, irregular wavelet frames have recently come to the forefront of current research due to questions concerning the robustness and stability of wavelet algorithms. A major difficulty in the study of these systems is the highly sensitive interplay between geometric properties of a sequence of time-scale indices and frame properties of the associated wavelet systems. This volume provides the first thorough and comprehensive treatment of irregular wavelet frames by introducing and employing a new notion of affine density as a highly effective tool for examining the geometry of sequences of time-scale indices. Many of the results are new and published for the first time. Topics include: qualitative and quantitative density conditions for existence of irregular wavelet frames, non-existence of irregular co-affine frames, the Nyquist phenomenon for wavelet systems, and approximation properties of irregular wavelet frames.

  3. Inhomogeneous self-affine carpets

    OpenAIRE

    Fraser, Jonathan M.

    2013-01-01

    We investigate the dimension theory of inhomogeneous self-affine carpets. Through the work of Olsen, Snigireva and Fraser, the dimension theory of inhomogeneous self-similar sets is now relatively well-understood, however, almost no progress has been made concerning more general non-conformal inhomogeneous attractors. If a dimension is countably stable, then the results are immediate and so we focus on the upper and lower box dimensions and compute these explicitly for large classes of inhomo...

  4. Alternative affinity tools: more attractive than antibodies?

    NARCIS (Netherlands)

    Ruigrok, V.J.B.; Levisson, M.; Eppink, M.H.M.; Smidt, H.; Oost, van der J.

    2011-01-01

    Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids

  5. Manifolds with integrable affine shape operator

    Directory of Open Access Journals (Sweden)

    Daniel A. Joaquín

    2005-05-01

    Full Text Available This work establishes the conditions for the existence of vector fields with the property that theirs covariant derivative, with respect to the affine normal connection, be the affine shape operatorS in hypersurfaces. Some results are obtained from this property and, in particular, for some kind of affine decomposable hypersurfaces we explicitely get the actual vector fields.

  6. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    Science.gov (United States)

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  7. Development of an aptamer-based affinity purification method for vascular endothelial growth factor

    Directory of Open Access Journals (Sweden)

    Maren Lönne

    2015-12-01

    Full Text Available Since aptamers bind their targets with high affinity and specificity, they are promising alternative ligands in protein affinity purification. As aptamers are chemically synthesized oligonucleotides, they can be easily produced in large quantities regarding GMP conditions allowing their application in protein production for therapeutic purposes. Several advantages of aptamers compared to antibodies are described in general within this paper. Here, an aptamer directed against the human Vascular Endothelial Growth Factor (VEGF was used as affinity ligand for establishing a purification platform for VEGF in small scale. The aptamer was covalently immobilized on magnetic beads in a controlled orientation resulting in a functional active affinity matrix. Target binding was optimized by introduction of spacer molecules and variation of aptamer density. Further, salt-induced target elution was demonstrated as well as VEGF purification from a complex protein mixture proving the specificity of protein-aptamer binding.

  8. Characterization of methacrylate chromatographic monoliths bearing affinity ligands.

    Science.gov (United States)

    Černigoj, Urh; Vidic, Urška; Nemec, Blaž; Gašperšič, Jernej; Vidič, Jana; Lendero Krajnc, Nika; Štrancar, Aleš; Podgornik, Aleš

    2016-09-16

    We investigated effect of immobilization procedure and monolith structure on chromatographic performance of methacrylate monoliths bearing affinity ligands. Monoliths of different pore size and various affinity ligands were prepared and characterized using physical and chromatographic methods. When testing protein A monoliths with different protein A ligand densities, a significant nonlinear effect of ligand density on dynamic binding capacity (DBC) for IgG was obtained and accurately described by Langmuir isotherm curve enabling estimation of protein A utilization as a function of ligand density. Maximal IgG binding capacity was found to be at least 12mg/mL exceeding theoretical monolayer adsorption value of 7.8mg/mL assuming hexagonal packing and IgG hydrodynamic diameter of 11nm. Observed discrepancy was explained by shrinkage of IgG during adsorption on protein A experimentally determined through calculated adsorbed IgG layer thickness of 5.4nm from pressure drop data. For monoliths with different pore size maximal immobilized densities of protein A as well as IgG dynamic capacity linearly correlates with monolith surface area indicating constant ligand utilization. Finally, IgGs toward different plasma proteins were immobilized via the hydrazide coupling chemistry to provide oriented immobilization. DBC was found to be flow independent and was increasing with the size of bound protein. Despite DBC was lower than IgG capacity to immobilized protein A, ligand utilization was higher. PMID:27554023

  9. Evaluation of ligand-binding affinity using polynomial empirical scoring functions.

    Science.gov (United States)

    de Azevedo, Walter Filgueira; Dias, Raquel

    2008-10-15

    Assessing protein-ligand interaction is of great importance for virtual screening initiatives in order to discover new drugs. The present work describes a set of empirical scoring functions to assess the binding affinity, involving terms for intermolecular hydrogen bonds and contact surface. The results show that our methodology works better to predict protein-ligand affinity when compared with XSCORE, a popular empirical scoring function.

  10. Rational self-affine tiles

    CERN Document Server

    Steiner, Wolfgang

    2012-01-01

    An integral self-affine tile is the solution of a set equation $\\mathbf{A} \\mathcal{T} = \\bigcup_{d \\in \\mathcal{D}} (\\mathcal{T} + d)$, where $\\mathbf{A}$ is an $n \\times n$ integer matrix and $\\mathcal{D}$ is a finite subset of $\\mathbb{Z}^n$. In the recent decades, these objects and the induced tilings have been studied systematically. We extend this theory to matrices $\\mathbf{A} \\in \\mathbb{Q}^{n \\times n}$. We define rational self-affine tiles as compact subsets of the open subring $\\mathbb{R}^n\\times \\prod_\\mathfrak{p} K_\\mathfrak{p}$ of the ad\\'ele ring $\\mathbb{A}_K$, where the factors of the (finite) product are certain $\\mathfrak{p}$-adic completions of a number field $K$ that is defined in terms of the characteristic polynomial of $\\mathbf{A}$. Employing methods from classical algebraic number theory, Fourier analysis in number fields, and results on zero sets of transfer operators, we establish a general tiling theorem for these tiles. We also associate a second kind of tiles with a rational matr...

  11. Graded electron affinity electron source

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, J.L.; Gray, H.F.; Jensen, K.L.; Jung, T.M. [Naval Research Laboratory, Washington, DC 20375 (United States)

    1996-05-01

    We describe a new electron source using electric field and low electron affinity semiconductor materials to bring charge to potential energy levels near the vacuum level while still in the solid. The basic idea involves moving some of the potential barrier from the surface to the bulk, and distributing the barrier over a thin layer below the surface. In so doing, the emission physics is changed fundamentally from a quantum mechanical tunneling process largely controlled by surface properties to a classical transport process largely controlled by the band structure of a wide bandgap semiconductor. The composition of the thin layer below the surface would be graded such that the conduction band minimum changes from an energy close to the substrate Fermi level to an energy significantly closer to the vacuum level. Electrons from the substrate would be drawn into the graded composition layer with an electric field produced by a pointed emitter structure and extraction gate similar to that used in field emitter arrays. Relative to a conventional field emitter array, the new source is expected to require lower extraction fields and exhibit improved emission uniformity, stability, and noise. The {ital I}{endash}{ital V} characteristics are not Fowler{endash}Nordheim and may include a saturation effect at high current densities that could be engineered to improve reliability and uniformity. The group III nitrides can be grown with a continuous range of composition that provides the range of electron affinity needed to produce the proposed electronic structure. {copyright} {ital 1996 American Vacuum Society}

  12. The affine quantum gravity programme

    International Nuclear Information System (INIS)

    The central principle of affine quantum gravity is securing and maintaining the strict positivity of the matrix { g-hat ab(x)} composed of the spatial components of the local metric operator. On spectral grounds, canonical commutation relations are incompatible with this principle, and they must be replaced by noncanonical, affine commutation relations. Due to the partial second-class nature of the quantum gravitational constraints, it is advantageous to use the recently developed projection operator method, which treats all quantum constraints on an equal footing. Using this method, enforcement of regularized versions of the gravitational operator constraints is formulated quite naturally by means of a novel and relatively well-defined functional integral involving only the same set of variables that appears in the usual classical formulation. It is anticipated that skills and insight to study this formulation can be developed by studying special, reduced-variable models that still retain some basic characteristics of gravity, specifically a partial second-class constraint operator structure. Although perturbatively nonrenormalizable, gravity may possibly be understood nonperturbatively from a hard-core perspective that has proved valuable for specialized models. Finally, developing a procedure to pass to the genuine physical Hilbert space involves several interconnected steps that require careful coordination

  13. The CRAPome: a contaminant repository for affinity purification–mass spectrometry data

    NARCIS (Netherlands)

    Mellacheruvu, D.; Wright, Z.; Couzens, A.L.; Low, T.Y.; Halim, V.A.; Heck, A.J.R.; Mohammed, S.; Nesvizhskii, A.

    2013-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background

  14. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    OpenAIRE

    Renata Angeli; da Paz, Nathalia V. N.; Maciel, Jackeline C.; Flávia F. B. Araújo; Paiva, Patrícia M. G.; Glícia M. T. Calazans; Ana Paula Valente; Almeida, Fábio C. L.; Coelho, Luana C. B. B.; Carvalho, Luiz B; Silva, Maria da Paz C.; Maria Tereza dos Santos Correia

    2009-01-01

    A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammon...

  15. Con A affinity glycoproteomics of normal human liver tissue

    Institute of Scientific and Technical Information of China (English)

    SUN QiangLing; LU HaoJie; LIU YinKun; LU WenJing; CHENG Gang; ZHOU HaiJun; ZHOU XinWen; WEI LiMing; DAI Zhi; GUO Kun

    2007-01-01

    In order to establish the novel high throughput, high efficiency and Iow cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.

  16. Con A affinity glycoproteomics of normal human liver tissue

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were gener- ated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence stain- ing based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were de- tected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF- MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.

  17. Methods for Improving Aptamer Binding Affinity

    OpenAIRE

    Hijiri Hasegawa; Nasa Savory; Koichi Abe; Kazunori Ikebukuro

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of a...

  18. Ordinary differential equations in affine geometry

    Directory of Open Access Journals (Sweden)

    Salvador Gigena

    1996-05-01

    Full Text Available The method of qualitative analysis is used, as applied to a class of fourth order, nonlinear ordinary differential equations, in order to classify, both locally and globally, two classes of hypersurfaces of decomposable type in affine geometry: those with constant unimodular affine mean curvature L , and those with constant Riemannian scalar curvature R. This allows to provide a large number of new examples of hypersurfaces in affine geometry.

  19. Ordinary differential equations in affine geometry

    OpenAIRE

    Salvador Gigena

    1996-01-01

    The method of qualitative analysis is used, as applied to a class of fourth order, nonlinear ordinary differential equations, in order to classify, both locally and globally, two classes of hypersurfaces of decomposable type in affine geometry: those with constant unimodular affine mean curvature L , and those with constant Riemannian scalar curvature R. This allows to provide a large number of new examples of hypersurfaces in affine geometry.

  20. Affine connections, midpoint formation, and point reflection

    DEFF Research Database (Denmark)

    Kock, Anders

    2011-01-01

    We describe some differential-geometric structures in combinatorial terms: namely affine connections and their torsion and curvature, and we show that torsion free affine connections may equivalently be presented in terms of some simpler combinatorial structure: midpoint formation, and point refl...... reflection (geodesic symmetry). The method employed is that of synthetic differential geometry, which is briefly explained.......We describe some differential-geometric structures in combinatorial terms: namely affine connections and their torsion and curvature, and we show that torsion free affine connections may equivalently be presented in terms of some simpler combinatorial structure: midpoint formation, and point...

  1. A Novel Vertex Affinity for Community Detection

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Andy [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Sanders, Geoffrey [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Henson, Van [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vassilevski, Panayot [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  2. A High-Affinity Metal-Binding Peptide From Escherichia Coli Hypb

    Energy Technology Data Exchange (ETDEWEB)

    Chung, K.C.Chan; Cao, L.; Dias, A.V.; Pickering, I.J.; George, G.N.; Zamble, D.B.

    2009-05-12

    The high-affinity nickel-binding site of the Escherichia coli [NiFe]-hydrogenase accessory protein HypB was localized to residues at the immediate N-terminus of the protein. Modification of a metal-binding fusion protein, site-directed mutagenesis experiments, and DFT calculations were used to identify the N-terminal amine as a ligand as well as the three cysteine residues in the CXXCGCXXX motif. This sequence can be removed from the protein and both a synthesized peptide and a protein fusion bind nickel with a similar affinity and the same structure as the parent metalloprotein, indicating the self-sufficiency of this high-affinity nickel-binding sequence.

  3. 棉铃虫中肠氨肽酶 APN4与 Cry1Ac、Cry2Aa 结合能力的比较%Comparison of the binding affinity of the APN4 receptor in Helicoverpa armigera (Hübner) to the Cry1Ac and Cry2Aa insecticidal proteins

    Institute of Scientific and Technical Information of China (English)

    袁向东; 葛朝虹; 肖玉涛; 梁革梅

    2015-01-01

    Objectives] Bt (Bacillus thuringiensis) insecticidal proteins have been widely adopted to control agricultural pests because of their high target specificity. The binding of a Bt insecticidal protein to its specific receptor in the insect midgut plays a key role in the insecticidal action process. Aminopeptidase N (APN) is one of the major Bt protein receptors. To better characterize the molecular mechanism underlying the insecticidal activity of different Bt insecitcidal proteins, and lay the foundation for Bt resistance management and novel Bt insecticidal protein development, we analyzed the binding affinity of aminopeptidase N4 (APN4) to the Cry1Ac and Cry2Aa insecitcidal proteins in Helicoverpa armigera. [Methods] The binding affinity of aminopeptidase N4 (APN4) was assessed using ligand blot analysis and an ELISA binding assay, respectively. [Results] The results show that recombinant APN4 could bind to both Cry1Ac and Cry2A; their respective dissociation constants were 46.7 nmol/L and 26.5 nmol/L. [Conclusion] The results suggest that there was no significant difference in the binding affinity of APN4 to Cry1Ac and Cry2Aa in H. armigera.%【目的】 Bt 杀虫蛋白(Bacillus thuringiensis)具有高度的靶标特异性,已经被广泛用于农业害虫防治。Bt 杀虫蛋白要发挥杀虫活性,必须首先与其受体蛋白结合,氨肽酶 N(Aminopeptidase N)是一类重要的 Bt 受体蛋白。因此,分析该受体与 Bt 杀虫蛋白的结合能力,可为进一步明确不同 Bt 的分子作用机制、Bt 的抗性治理以及新 Bt 的开发应用等提供借鉴。【方法】本文利用 Ligand blot 和 Elisa 方法比较了棉铃虫 Helicoverpa armigera 中肠 APN4(Aminopeptidase N4,APN4)与 Cry1Ac、Cry2Aa 的结合能力。【结果】原核表达的 APN4片段与活化的 Cry1Ac、Cry2Aa 都可以结合,解离常数(Kd)分别是48.59 nmol/L和21.73 nmol/L。【结论】 APN4片段与 Cry1Ac、Cry2Aa 的结合能力

  4. Affine processes on positive semidefinite matrices

    CERN Document Server

    Cuchiero, Christa; Mayerhofer, Eberhard; Teichmann, Josef

    2009-01-01

    This paper provides the mathematical foundation for stochastically continuous affine processes on the cone of positive semidefinite symmetric matrices. These matrix-valued affine processes have arisen from a large and growing range of useful applications in finance, including multi-asset option pricing with stochastic volatility and correlation structures, and fixed-income models with stochastically correlated risk factors and default intensities.

  5. Lectures on extended affine Lie algebras

    CERN Document Server

    Neher, Erhard

    2010-01-01

    We give an introduction to the structure theory of extended affine Lie algebras, which provide a common framework for finite-dimensional semisimple, affine and toroidal Lie algebras. The notes are based on a lecture series given during the Fields Institute summer school at the University of Ottawa in June 2009.

  6. Global affine differential geometry of hypersurfaces

    CERN Document Server

    Li, An-Min; Zhao, Guosong; Hu, Zejun

    2015-01-01

    This book draws a colorful and widespread picture of global affine hypersurface theory up to the most recent state. Moreover, the recent development revealed that affine differential geometry- as differential geometry in general- has an exciting intersection area with other fields of interest, like partial differential equations, global analysis, convex geometry and Riemann surfaces.

  7. Dyes with high affinity for polylactide

    Institute of Scientific and Technical Information of China (English)

    Liang He; Shu Fen Zhang; Bing Tao Tang; Li Li Wang; Jin Zong Yang

    2007-01-01

    Attempts were made to develop dyes with high affinity for polylactide as an alternative to the existent commercial disperse dyes.The dyes synthesized according to the affinity concept of dye to polylactide exhibited excellent dyeing properties on polylactide compared with the commercial disperse dyes.

  8. Free C+ actions on affine threefolds

    OpenAIRE

    Kraft, H.

    2005-01-01

    We study algebraic actions of the additive group C+ on an affine threefold X and prove a smoothness property for the quotient morphism X -< X//C+. Then, following Shulim Kaliman, we give a proof of the conjecture that every free C+ action on affine 3-space C^3 is a translation.

  9. Porosity of Self-affine Sets

    Institute of Scientific and Technical Information of China (English)

    Lifeng XI

    2008-01-01

    In this paper,it is proved that any self-affine set satisfying the strong separation condition is uniformly porous.The author constructs a self-affine set which is not porous,although the open set condition holds.Besides,the author also gives a C1 iterated function system such that its invariant set is not porous.

  10. On affine non-negative matrix factorization

    DEFF Research Database (Denmark)

    Laurberg, Hans; Hansen, Lars Kai

    2007-01-01

    We generalize the non-negative matrix factorization (NMF) generative model to incorporate an explicit offset. Multiplicative estimation algorithms are provided for the resulting sparse affine NMF model. We show that the affine model has improved uniqueness properties and leads to more accurate...

  11. Phosphopeptide enrichment by immobilized metal affinity chromatography

    DEFF Research Database (Denmark)

    Thingholm, Tine E.; Larsen, Martin R.

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...

  12. Improving image segmentation by learning region affinities

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lakshman [Los Alamos National Laboratory; Yang, Xingwei [TEMPLE UNIV.; Latecki, Longin J [TEMPLE UNIV.

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  13. Reflectable bases for affine reflection systems

    CERN Document Server

    Azam, Saeid; Yousofzadeh, Malihe

    2011-01-01

    The notion of a "root base" together with its geometry plays a crucial role in the theory of finite and affine Lie theory. However, it is known that such a notion does not exist for the recent generalizations of finite and affine root systems such as extended affine root systems and affine reflection systems. As an alternative, we introduce the notion of a "reflectable base", a minimal subset $\\Pi$ of roots such that the non-isotropic part of the root system can be recovered by reflecting roots of $\\Pi$ relative to the hyperplanes determined by $\\Pi$. We give a full characterization of reflectable bases for tame irreducible affine reflection systems of reduced types, excluding types $E_{6,7,8}$. As a byproduct of our results, we show that if the root system under consideration is locally finite then any reflectable base is an integral base.

  14. Fatty acid and drug binding to a low-affinity component of human serum albumin, purified by affinity chromatography

    DEFF Research Database (Denmark)

    Vorum, H; Pedersen, A O; Honoré, B

    1992-01-01

    of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid...... and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture...

  15. Novel trends in affinity biosensors: current challenges and perspectives

    International Nuclear Information System (INIS)

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives. (topical review)

  16. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype

    DEFF Research Database (Denmark)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte;

    2002-01-01

    with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable......Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor...... Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding...

  17. Non-Zenoness of piecewise affine dynamical systems and affine complementarity systems with inputs

    Institute of Scientific and Technical Information of China (English)

    Le Quang THUAN

    2014-01-01

    In the context of continuous piecewise affine dynamical systems and affine complementarity systems with inputs, we study the existence of Zeno behavior, i.e., infinite number of mode transitions in a finite-length time interval, in this paper. The main result reveals that continuous piecewise affine dynamical systems with piecewise real-analytic inputs do not exhibit Zeno behavior. Applied the achieved result to affine complementarity systems with inputs, we also obtained a similar conclusion. A direct benefit of the main result is that one can apply smooth ordinary differential equations theory in a local manner for the analysis of continuous piecewise affine dynamical systems with inputs.

  18. An Activation Force-based Affinity Measure for Analyzing Complex Networks

    OpenAIRE

    Jun Guo; Hanliang Guo; Zhanyi Wang

    2011-01-01

    Affinity measure is a key factor that determines the quality of the analysis of a complex network. Here, we introduce a type of statistics, activation forces, to weight the links of a complex network and thereby develop a desired affinity measure. We show that the approach is superior in facilitating the analysis through experiments on a large-scale word network and a protein-protein interaction (PPI) network consisting of ∼5,000 human proteins. The experiment on the word network verifies tha...

  19. Blind prediction of host-guest binding affinities: A new SAMPL3 challenge

    OpenAIRE

    Muddana, Hari S.; Varnado, C. Daniel; Bielawski, Christopher W.; Urbach, Adam R.; Isaacs, Lyle; Geballe, Matthew T; Gilson, Michael K.

    2012-01-01

    The computational prediction of protein-ligand binding affinities is of central interest in early-stage drug-discovery, and there is a widely recognized need for improved methods. Low molecular weight receptors and their ligands—i.e. host-guest systems – represent valuable test-beds for such affinity prediction methods, because their small size makes for fast calculations and relatively facile numerical convergence. The SAMPL3 community exercise included the first ever blind prediction challe...

  20. Calculation of Host-Guest Binding Affinities Using a Quantum-Mechanical Energy Model

    OpenAIRE

    Muddana, Hari S.; Gilson, Michael K.

    2012-01-01

    The prediction of protein-ligand binding affinities is of central interest in computer-aided drug discovery, but it is still difficult to achieve a high degree of accuracy. Recent studies suggesting that available force fields may be a key source of error motivate the present study, which reports the first mining minima (M2) binding affinity calculations based on a quantum mechanical energy model, rather than an empirical force field. We apply a semi-empirical quantum-mechanical energy functi...

  1. Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and Fus3 with the upstream MAP kinase kinase Ste7.

    OpenAIRE

    Bardwell, L; Cook, J G; E. C. Chang; Cairns, B R; Thorner, J

    1996-01-01

    Kss1 and Fus3 are mitogen-activated protein kinases (MAPKs or ERKs), and Ste7 is their activating MAPK/ERK kinase (MEK), in the pheromone response pathway of Saccharomyces cerevisiae. To investigate the potential role of specific interactions between these enzymes during signaling, their ability to associate with each other was examined both in solution and in vivo. When synthesized by in vitro translation, Kss1 and Fus3 could each form a tight complex (Kd of approximately 5 nM) with Ste7 in ...

  2. Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

    Science.gov (United States)

    Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2011-05-01

    Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625

  3. Protein-Protein Interaction Detection: Methods and Analysis

    OpenAIRE

    V. Srinivasa Rao; Srinivas, K.; Sujini, G. N.; G. N. Sunand Kumar

    2014-01-01

    Protein-protein interaction plays key role in predicting the protein function of target protein and drug ability of molecules. The majority of genes and proteins realize resulting phenotype functions as a set of interactions. The in vitro and in vivo methods like affinity purification, Y2H (yeast 2 hybrid), TAP (tandem affinity purification), and so forth have their own limitations like cost, time, and so forth, and the resultant data sets are noisy and have more false positives to annotate t...

  4. Affinity Proteomics in the mountains: Alpbach 2015.

    Science.gov (United States)

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  5. Affinization of category O for quantum groups

    CERN Document Server

    Young, C A S

    2012-01-01

    Let g be a simple Lie algebra. We consider the category O-hat of those modules over the affine quantum group Uq(g-hat) whose Uq(g)-weights have finite multiplicity and lie in a finite union of cones generated by negative roots. We show that many properties of the category of the finite-dimensional representations naturally extend to the category O-hat. In particular, we develop the theory of q-characters and define the minimal affinizations of parabolic Verma modules. In types ABCFG we classify these minimal affinizations and conjecture a Weyl denominator type formula for their characters.

  6. Corner Transfer Matrices and Quantum Affine Algebras

    CERN Document Server

    Foda, O E; Foda, Omar; Miwa, Tetsuji

    1992-01-01

    Let H be the corner-transfer-matrix Hamiltonian for the six-vertex model in the anti-ferroelectric regime. It acts on the infinite tensor product W = V . V . V ....., where is the 2-dimensional irreducible representation of the quantum affine sl(2). We observe that H is the derivation of quantum affine sl(2), and conjecture that the eigenvectors of H form the level-1 vacuum representation of quantum affine sl(2). We report on checks in support of our conjecture.

  7. On the Meaning of Affinity Limits in B-Cell Epitope Prediction for Antipeptide Antibody-Mediated Immunity

    Directory of Open Access Journals (Sweden)

    Salvador Eugenio C. Caoili

    2012-01-01

    Full Text Available B-cell epitope prediction aims to aid the design of peptide-based immunogens (e.g., vaccines for eliciting antipeptide antibodies that protect against disease, but such antibodies fail to confer protection and even promote disease if they bind with low affinity. Hence, the Immune Epitope Database (IEDB was searched to obtain published thermodynamic and kinetic data on binding interactions of antipeptide antibodies. The data suggest that the affinity of the antibodies for their immunizing peptides appears to be limited in a manner consistent with previously proposed kinetic constraints on affinity maturation in vivo and that cross-reaction of the antibodies with proteins tends to occur with lower affinity than the corresponding reaction of the antibodies with their immunizing peptides. These observations better inform B-cell epitope prediction to avoid overestimating the affinity for both active and passive immunization; whereas active immunization is subject to limitations of affinity maturation in vivo and of the capacity to accumulate endogenous antibodies, passive immunization may transcend such limitations, possibly with the aid of artificial affinity-selection processes and of protein engineering. Additionally, protein disorder warrants further investigation as a possible supplementary criterion for B-cell epitope prediction, where such disorder obviates thermodynamically unfavorable protein structural adjustments in cross-reactions between antipeptide antibodies and proteins.

  8. Automorphisms in Birational and Affine Geometry

    CERN Document Server

    Ciliberto, Ciro; Flenner, Hubert; McKernan, James; Prokhorov, Yuri; Zaidenberg, Mikhail

    2014-01-01

    The main focus of this volume is on the problem of describing the automorphism groups of affine and projective varieties, a classical subject in algebraic geometry where, in both cases, the automorphism group is often infinite dimensional. The collection covers a wide range of topics and is intended for researchers in the fields of classical algebraic geometry and birational geometry (Cremona groups) as well as affine geometry with an emphasis on algebraic group actions and automorphism groups. It presents original research and surveys and provides a valuable overview of the current state of the art in these topics. Bringing together specialists from projective, birational algebraic geometry and affine and complex algebraic geometry, including Mori theory and algebraic group actions, this book is the result of ensuing talks and discussions from the conference “Groups of Automorphisms in Birational and Affine Geometry” held in October 2012, at the CIRM, Levico Terme, Italy. The talks at the conference high...

  9. Candidate SNP Markers of Gender-Biased Autoimmune Complications of Monogenic Diseases Are Predicted by a Significant Change in the Affinity of TATA-Binding Protein for Human Gene Promoters

    Science.gov (United States)

    Ponomarenko, Mikhail P.; Arkova, Olga; Rasskazov, Dmitry; Ponomarenko, Petr; Savinkova, Ludmila; Kolchanov, Nikolay

    2016-01-01

    Some variations of human genome [for example, single nucleotide polymorphisms (SNPs)] are markers of hereditary diseases and drug responses. Analysis of them can help to improve treatment. Computer-based analysis of millions of SNPs in the 1000 Genomes project makes a search for SNP markers more targeted. Here, we combined two computer-based approaches: DNA sequence analysis and keyword search in databases. In the binding sites for TATA-binding protein (TBP) in human gene promoters, we found candidate SNP markers of gender-biased autoimmune diseases, including rs1143627 [cachexia in rheumatoid arthritis (double prevalence among women)]; rs11557611 [demyelinating diseases (thrice more prevalent among young white women than among non-white individuals)]; rs17231520 and rs569033466 [both: atherosclerosis comorbid with related diseases (double prevalence among women)]; rs563763767 [Hughes syndrome-related thrombosis (lethal during pregnancy)]; rs2814778 [autoimmune diseases (excluding multiple sclerosis and rheumatoid arthritis) underlying hypergammaglobulinemia in women]; rs72661131 and rs562962093 (both: preterm delivery in pregnant diabetic women); and rs35518301, rs34166473, rs34500389, rs33981098, rs33980857, rs397509430, rs34598529, rs33931746, rs281864525, and rs63750953 (all: autoimmune diseases underlying hypergammaglobulinemia in women). Validation of these predicted candidate SNP markers using the clinical standards may advance personalized medicine. PMID:27092142

  10. The state of the art of removal of prion proteins in SD-FFP, by specific prion affinity chromatography and its impact on the hemostatic characteristics of the product.

    Science.gov (United States)

    Neisser-Svae, Andrea; Seghatchian, Jerard

    2015-04-01

    In recent Coimbra' Conference, on the pre-launch of pathogen reduced-FFP for the local clinical use, the question was raised, by the moderator, on the efficacy of the current methodology used for prion removal processes and its influence on the overall quality and safety of the final product. This brief paper put together by speaker of this session and the moderator, as a consensus of opinions, which was largely discussed during Q&A session, to make it available to a large group of readers of transfusion apheresis science, who might be interested to this topic. In short the capacity of the current process of Octaplas to remove prion is in order of 5.6 log10/ID50 reduction based on several animal studies. Moreover the changes in coagulation and inhibitors are within acceptable range and bioequivalent to untreated FFP with no sign of inferiority. This paper describes in brief a technology update on solvent/detergent treated plasma, an alternative to FFP but with increased pathogen safety. The biochemical profile of the final product is comparable with FFP and contains all clinically relevant plasma proteins. Furthermore, Octaplas is a product that, in long term, reduces health care costs.

  11. Synthesis of a New Series of Bone Affinity Compounds

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A new series of bone affinity compounds were synthesized by linking chrysophanol with 5-fluorouracil derivatives. Their bone affinity was established by hydroxyapafive (HA)affinity experiment in vitro, and their cytostatic effects were shown by the MTT assay.

  12. A MEMS Dielectric Affinity Glucose Biosensor

    OpenAIRE

    Xian HUANG; Li, SiQi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-01-01

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concent...

  13. On Affine Fusion and the Phase Model

    OpenAIRE

    Walton, Mark A.

    2012-01-01

    A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the $su(n)$ Wess-Zumino-Novikov-Witten (WZNW) conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connec...

  14. Purely affine elementary su(N) fusions

    OpenAIRE

    Rasmussen, Jorgen; Walton, Mark A.

    2001-01-01

    We consider three-point couplings in simple Lie algebras -- singlets in triple tensor products of their integrable highest weight representations. A coupling can be expressed as a linear combination of products of finitely many elementary couplings. This carries over to affine fusion, the fusion of Wess-Zumino-Witten conformal field theories, where the expressions are in terms of elementary fusions. In the case of su(4) it has been observed that there is a purely affine elementary fusion, i.e...

  15. Complete algebraic vector fields on affine surfaces

    OpenAIRE

    Kaliman, Shulim; Kutzschebauch, Frank; Leuenberger, Matthias

    2014-01-01

    Let $\\AAutH (X)$ be the subgroup of the group $\\AutH (X)$ of holomorphic automorphisms of a normal affine algebraic surface $X$ generated by elements of flows associated with complete algebraic vector fields. Our main result is a classification of all normal affine algebraic surfaces $X$ quasi-homogeneous under $\\AAutH (X)$ in terms of the dual graphs of the boundaries $\\bX \\setminus X$ of their SNC-completions $\\bX$.

  16. Fan affinity laws from a collision model

    International Nuclear Information System (INIS)

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour of air is incorporated. Our calculations prove the affinity laws and provide numerical estimates of the air delivery, thrust and drag on a rotating fan. (paper)

  17. Cellulose binding domain proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc (Davis, CA); Doi, Roy (Davis, CA)

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  18. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    Science.gov (United States)

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  19. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies.

    Science.gov (United States)

    Julian, Mark C; Lee, Christine C; Tiller, Kathryn E; Rabia, Lilia A; Day, Evan K; Schick, Arthur J; Tessier, Peter M

    2015-10-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33-42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. PMID:26386257

  20. Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes.

    Science.gov (United States)

    Jain, Tarun; Jayaram, B

    2007-06-01

    Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).

  1. Understanding proton affinity of tyrosine sidechain in hydrophobic confinement

    Indian Academy of Sciences (India)

    T G Abi; T Karmakar; S Taraphder

    2012-01-01

    Tyrosine is an important amino acid residue that plays a key role in several biochemical transformations such as, abstraction/donation of proton from/by its sidechain. We present here a density functional study on the proton affinity of tyrosine sidechain suspended inside the core of a single walled carbon nanotube that mimics the environment of protein structural pores and molecular channels. Tyrosine is found to exhibit a lower reactivity on confinement and unlike several other polar amino acid sidechains, its reactivity does not respond to hydrogen bonding with neighbouring hydroxyl groups.

  2. Affine modifications and affine hypersurfaces with a very transitive automorphism group

    OpenAIRE

    Kaliman, Shulim; ZAIDENBERG, MIKHAIL

    1998-01-01

    We study a kind of modification of an affine domain which produces another affine domain. First appeared in passing in the basic paper of O. Zariski (1942), it was further considered by E.D. Davis (1967). The first named author applied its geometric counterpart to construct contractible smooth affine varieties non-isomorphic to Euclidean spaces. Here we provide certain conditions which guarantee preservation of the topology under a modification. As an application, we show that the group of bi...

  3. Agonist binding to high-affinity dopamine sites

    Energy Technology Data Exchange (ETDEWEB)

    Tedesco, J.L.

    1985-01-01

    The authors have characterized the dopamine D/sub 3/ site and its binding requirements. The dopamine D/sub 3/ site in calf caudate crude homogenate has a site density of 214-230 fmoles/mg. protein by both /sup 3/H-apomorphine (/sup 3/H-AOP) and /sup 3/H-dopamine (/sup 3/H-DA) Scatchard analysis of specific binding (SB). Stereospecific subsets of /sup 3/H-APO and /sup 3/H-DA sites were defined by the use of agonist and antagonist enantiomer-pairs as a rigorous test for D/sub 3/ site heterogeneity. IC/sub 50/ values for both /sup 3/H-APO and /sup 3/H-DA SB sites were assessed for 55 agonist ligands and an excellent correlation was obtained. The authors conclude that both /sup 3/H-ligands label the same D/sub 3/ site. The D/sub 3/ site affinities of 105 dopamine-agonist ligands, in particular 2-aminotetralins,, aporphines and flexible dopamine analogues were measured. Low D/sub 3/-site affinities of N-quaternary analogues confirm the need for a lone pair. Subadditivity of substituents' effects in semi-flexible DA analogues confirms their postulate that sidechain conformation is the critical determinant of affinity. They conclude that there are at least two high-affinity ligand conformations of the DA sidechain pharmacophore. These binding requirements are presented as two interface-Geometry tetrahedral models of the double H-bond interface between the D/sub 3/ site and the ideal ligand.

  4. [Protein expression and purification].

    Science.gov (United States)

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  5. 苹果果实细胞质中依赖活体组织的ABA高亲和力特异结合蛋白%In Vivo Tissue-dependent Abscisic Acid Specific-binding Proteins with High Affinity in Cytosol of Developing Apple Fruits

    Institute of Scientific and Technical Information of China (English)

    张大鹏; 陈尚武

    2001-01-01

    The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sit es localized in cytosol were identified and characterized in the flesh of develo ping apple (Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the s ubcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in 3H-ABA binding medium, the flesh tissue discs we re directly in vivo incubated in 3H-ABA binding medium, a high ABA bin ding activity to the cytosolic fraction isolated from these tissue discs was detect ed. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA bin ding needs a living state of tissue. The in vivo tissue-dependent binding si tes were shown to possess protein nature with both active serine residua and thiol-g roup of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting g ave evidence of two different classes of ABA binding proteins, one with a higher affinity (Kd=2.9 nmol/L) and the other with lower affinity (Kd=71.4 nmo l/L). Ph aseic acid, 2-trans-4-trans-ABA or cis-trans-(-)-ABA had substantia lly no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hyp othesized that the detected ABA-binding proteins may be putative ABA-receptors t hat mediate ABA signals during fruit development.%将苹果(Malus pumila L. cv. Starkrimon)果肉微粒体和细胞可溶组分在含有 3H-ABA的缓冲介质中分别温育,仅在细胞

  6. Secretion of Recombinant Proteins in Mammalian Cells Directed by Growth Hormone Signal Peptide%生长激素信号肽可诱导重组蛋白外分泌表达

    Institute of Scientific and Technical Information of China (English)

    张志谦; 李金萍; 胡颖

    2005-01-01

    Signal peptide capable of efficiently directing many protein secretion in mammalian cells is one ot the key elements in recombinant protein production, gene therapy and the development of DNA vaccines. In order to explore the possibility of rat growth hormone signal peptide as such an element, a new vector based on the mammalian expression vector pcDNA3 was constructed by employing rat growth hormone (rGH) signal peptide as leading sequence, followed by multiple cloning sites, the myc epitope-tag and 6 × his purification tag in the expression cassette. The vector was validated by successfully expressing and secretion of chick MMP-2 Cterminal PEX domain, a potential angiogenesis inhibitor, and tandem peptide repeats of myc epitope-tag in COS-7 cells. These results suggest that rat growth hormone signal peptide is effective in the mediation of recombinant protein expression and secretion, and this vector provides a new tool for universal cloning and secretion of exogenous proteins in mammalian cells.

  7. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    Science.gov (United States)

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  8. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    Science.gov (United States)

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media.

  9. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    Science.gov (United States)

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  10. Classification of neocortical interneurons using affinity propagation

    Science.gov (United States)

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  11. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    Science.gov (United States)

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  12. On Affine Fusion and the Phase Model

    Directory of Open Access Journals (Sweden)

    Mark A. Walton

    2012-11-01

    Full Text Available A brief review is given of the integrable realization of affine fusion discovered recently by Korff and Stroppel. They showed that the affine fusion of the su(n Wess-Zumino-Novikov-Witten (WZNW conformal field theories appears in a simple integrable system known as the phase model. The Yang-Baxter equation leads to the construction of commuting operators as Schur polynomials, with noncommuting hopping operators as arguments. The algebraic Bethe ansatz diagonalizes them, revealing a connection to the modular S matrix and fusion of the su(n WZNW model. The noncommutative Schur polynomials play roles similar to those of the primary field operators in the corresponding WZNW model. In particular, their 3-point functions are the su(n fusion multiplicities. We show here how the new phase model realization of affine fusion makes obvious the existence of threshold levels, and how it accommodates higher-genus fusion.

  13. Affinity chromatography of bacterial lactate dehydrogenases.

    Science.gov (United States)

    Kelly, N; Delaney, M; O'Carra, P

    1978-06-01

    The affinity system used was the immobilized oxamate derivative previously used to purify mammalian lactate dehydrogenases. The bacterial dehydrogenases specific for the L-stereoisomer of lactate behaved in the same way as the mammalian enzymes, binding strongly in the presence of NADH. The D-lactate-specific enzymes, however, did not show any biospecific affinity for this gel. The L-specific enzymes could be purified to homogeneity in one affinity-chromatographic step. The D-specific enzymes could be efficiently separated from the L-specific ones and could then be further purified on an immobilized NAD derivative. The mechanism of activation of the lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate was investigated by using the immobilized oxamate gel. PMID:666726

  14. The Affine q-Schur algebra

    OpenAIRE

    Green, R. M.

    1997-01-01

    We introduce an analogue of the $q$-Schur algebra associated to Coxeter systems of type $\\hat A_{n-1}$. We give two constructions of this algebra. The first construction realizes the algebra as a certain endomorphism algebra arising from an affine Hecke algebra of type $\\hat A_{r-1}$, where $n \\geq r$. This generalizes the original $q$-Schur algebra as defined by Dipper and James, and the new algebra contains the ordinary $q$-Schur algebra and the affine Hecke algebra as subalgebras. Using th...

  15. Affine Projection Algorithm Using Regressive Estimated Error

    OpenAIRE

    Zhang, Shu; Zhi, Yongfeng

    2011-01-01

    An affine projection algorithm using regressive estimated error (APA-REE) is presented in this paper. By redefining the iterated error of the affine projection algorithm (APA), a new algorithm is obtained, and it improves the adaptive filtering convergence rate. We analyze the iterated error signal and the stability for the APA-REE algorithm. The steady-state weights of the APA-REE algorithm are proved to be unbiased and consist. The simulation results show that the proposed algorithm has a f...

  16. Control and estimation of piecewise affine systems

    CERN Document Server

    Xu, Jun

    2014-01-01

    As a powerful tool to study nonlinear systems and hybrid systems, piecewise affine (PWA) systems have been widely applied to mechanical systems. Control and Estimation of Piecewise Affine Systems presents several research findings relating to the control and estimation of PWA systems in one unified view. Chapters in this title discuss stability results of PWA systems, using piecewise quadratic Lyapunov functions and piecewise homogeneous polynomial Lyapunov functions. Explicit necessary and sufficient conditions for the controllability and reachability of a class of PWA systems are

  17. Affine Invariant Character Recognition by Progressive Removing

    Science.gov (United States)

    Iwamura, Masakazu; Horimatsu, Akira; Niwa, Ryo; Kise, Koichi; Uchida, Seiichi; Omachi, Shinichiro

    Recognizing characters in scene images suffering from perspective distortion is a challenge. Although there are some methods to overcome this difficulty, they are time-consuming. In this paper, we propose a set of affine invariant features and a new recognition scheme called “progressive removing” that can help reduce the processing time. Progressive removing gradually removes less feasible categories and skew angles by using multiple classifiers. We observed that progressive removing and the use of the affine invariant features reduced the processing time by about 60% in comparison to a trivial one without decreasing the recognition rate.

  18. Adsorption affinity of anions on metal oxyhydroxides

    Science.gov (United States)

    Pechenyuk, S. I.; Semushina, Yu. P.; Kuz'mich, L. F.

    2013-03-01

    The dependences of anion (phosphate, carbonate, sulfate, chromate, oxalate, tartrate, and citrate) adsorption affinity anions from geometric characteristics, acid-base properties, and complex forming ability are generalized. It is shown that adsorption depends on the nature of both the anions and the ionic medium and adsorbent. It is established that anions are generally grouped into the following series of adsorption affinity reduction: PO{4/3-}, CO{3/2-} > C2O{4/2-}, C(OH)(CH2)2(COO){3/3-}, (CHOH)2(COO){2/2-} > CrO{4/2-} ≫ SO{4/2-}.

  19. Periodic cyclic homology of affine Hecke algebras

    CERN Document Server

    Solleveld, Maarten

    2009-01-01

    This is the author's PhD-thesis, which was written in 2006. The version posted here is identical to the printed one. Instead of an abstract, the short list of contents: Preface 5 1 Introduction 9 2 K-theory and cyclic type homology theories 13 3 Affine Hecke algebras 61 4 Reductive p-adic groups 103 5 Parameter deformations in affine Hecke algebras 129 6 Examples and calculations 169 A Crossed products 223 Bibliography 227 Index 237 Samenvatting 245 Curriculum vitae 253

  20. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    Science.gov (United States)

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  1. Crossing Chris: Some Markerian Affinities

    Directory of Open Access Journals (Sweden)

    Adrian Martin

    2010-01-01

    -pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

    Abstract (E: This essay creatively explores a group of artists, writers, and other special individuals whose work or life story can be described as having an intriguing affinity with the protean career of Chris Marker. Avoiding the ‘usual suspects’ (such as Godard or Sebald, it discusses gossip columnist Milt Machlin, record collector Harry Smith, painter Gianfranco Baruchello, writer-filmmaker Edgardo Cozarinsky, and several others. From this constellation, a particular view of Markerian poetics emerges, touching upon the meanings of anonymity, storytelling, history and archiving.

     

    Abstract (F: Cet essai brosse de manière créative le portrait d’un groupe d'artistes, d'écrivains et d'autres personnes particulières dont le travail ou la biographie peuvent être décrits comme montrant une étrange mais certaine connivence avec la carrière protéiforme de Chris Marker. Evitant les lieux communs (comme Godard ou Sebald, cet article trace des références moins attendues :

  2. Fan Affinity Laws from a Collision Model

    Science.gov (United States)

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  3. Classification of neocortical interneurons using affinity propagation

    Directory of Open Access Journals (Sweden)

    Roberto eSantana

    2013-12-01

    Full Text Available In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. Neuronal classification has been a difficult problem because it is unclear what a neuronal cell class actually is and what are the best characteristics are to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological or molecular characteristics, when applied to selected datasets, have provided quantitative and unbiased identification of distinct neuronal subtypes. However, better and more robust classification methods are needed for increasingly complex and larger datasets. We explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. In fact, using a combined anatomical/physiological dataset, our algorithm differentiated parvalbumin from somatostatin interneurons in 49 out of 50 cases. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  4. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    Science.gov (United States)

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation. PMID:27367467

  5. Application of monolithic affinity HPLC column for rapid determination of malt glycoproteins

    OpenAIRE

    Benkovská, D. (Dagmar); Flodrová, D. (Dana); Bobálová, J. (Janette)

    2013-01-01

    The aim of this study was to optimize separation and enrichment of barley malt glycoproteins on a monolithic ConA affinity HPLC column. ConA-bound proteins were separated on SDS-PAGE and identified using MALDI-TOF/TOF MS after chymotryptic digestion. Our proteomic analysis allowed successful determination of several putative malt glycoproteins.

  6. Increased synthesis of heparin affin regulatory peptide in the perforant path lesioned mouse hippocampal formation

    DEFF Research Database (Denmark)

    Poulsen, F R; Lagord, C; Courty, J;

    2000-01-01

    Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is a developmentally regulated extracellular matrix protein that induces cell proliferation and promotes neurite outgrowth in vitro as well as pre- and postsynaptic developmental...

  7. Purification of infectious canine parvovirus from cell culture by affinity chromatography with monoclonal antibodies.

    NARCIS (Netherlands)

    J. Groen (Jan); N. Juntti; J.S. Teppema; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Ab); G.F. Rimmelzwaan (Guus)

    1987-01-01

    textabstractImmuno affinity chromatography with virus neutralizing monoclonal antibodies, directed to the haemagglutinating protein of canine parvovirus (CPV) was used to purify and concentrate CPV from infected cell culture. The procedure was monitored by testing the respective fractions in an infe

  8. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

    Science.gov (United States)

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-01-01

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

  9. Application of Ni(II-assisted peptide bond hydrolysis to non-enzymatic affinity tag removal.

    Directory of Open Access Journals (Sweden)

    Edyta Kopera

    Full Text Available In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His(6. This method is based on a highly specific Ni(II reaction with (S/TXHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

  10. Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

    Science.gov (United States)

    Paba, Jaime; Ricart, Carlos A O; Fontes, Wagner; Santana, Jaime M; Teixeira, Antonio R L; Marchese, Jason; Williamson, Brian; Hunt, Tony; Karger, Barry L; Sousa, Marcelo V

    2004-01-01

    Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes. PMID:15253433

  11. Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ.

    Directory of Open Access Journals (Sweden)

    Lorenzo Sborgi

    Full Text Available GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding. These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein

  12. Binding Affinity and Capacity for the Uremic Toxin Indoxyl Sulfate

    Directory of Open Access Journals (Sweden)

    Eric Devine

    2014-01-01

    Full Text Available Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i, increasing the sodium chloride (NaCl concentration to achieve a higher ionic strength; (ii, increasing the temperature; and (iii, dilution. The effects on the dissociation constant KD and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. KD increased linearly with the NaCl concentration between 0.15 (KD = 13.2 ± 3.7 µM and 0.75 M (KD = 56.2 ± 2.0 µM. Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.

  13. New Insights into Viral Architecture via Affine Extended Symmetry Groups

    Directory of Open Access Journals (Sweden)

    T. Keef

    2008-01-01

    Full Text Available Since the seminal work of Caspar and Klug on the structure of the protein containers that encapsulate and hence protect the viral genome, it has been recognized that icosahedral symmetry is crucial for the structural organization of viruses. In particular, icosahedral symmetry has been invoked in order to predict the surface structures of viral capsids in terms of tessellations or tilings that schematically encode the locations of the protein subunits in the capsids. Whilst this approach is capable of predicting the relative locations of the proteins in the capsids, a prediction on the relative sizes of different virus particles in a family cannot be made. Moreover, information on the full 3D structure of viral particles, including the tertiary structures of the capsid proteins and the organization of the viral genome within the capsid are inaccessible with their approach. We develop here a mathematical framework based on affine extensions of the icosahedral group that allows us to address these issues. In particular, we show that the relative radii of viruses in the family of Polyomaviridae and the material boundaries in simple RNA viruses can be determined with our approach. The results complement Caspar and Klug's theory of quasi-equivalence and provide details on virus structure that have not been accessible with previous methods, implying that icosahedral symmetry is more important for virus architecture than previously appreciated.

  14. Study on Removal of Bilirubin with Magnetic Affinity Separation Technique

    Institute of Scientific and Technical Information of China (English)

    张凤宝; 王淑兰; 徐辉; 张国亮

    2003-01-01

    An affinity adsorbent, Cibacron Blue 3GA immobilized magnetic polyvinyl alcohol (PVA) microspheres was used for bilirubin removal taking the advantage of easy separation of magnetic sorbent from the biosystem.Fe3 O4 superparamagnetic particles was synthesized with hydrothermal reaction of ferrous chloride (FeC12) and ferric chloride (FeCl3). Such magnetic particles are then encapsulated in biocompatible PVA to form magnetic polymer microspheres sized from 2 to 15 nm with hydroxyl groups on its surface. Cibacron Blue 3GA, a dye-ligand, was covalently coupled with the polyvinyl alcohol through the nucleophilic reaction between the chloride of its triazine ring and the hydroxyl groups of PVA molecules under alkaline condition. The affinity adsorbent carried 21.1μmol Cibacron Blue 3GA per gram magnetic polymer microspheres was used to remove unconjugated and conjugated bilirubin from the solution which was composed of bilirubin or bilirubin and protein. After the adsorption, the adsorbent loaded with bilirubin was removed easily in the magnetic field.

  15. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    Science.gov (United States)

    Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

  16. Impact of crystalline quality on neuronal affinity of pristine graphene.

    Science.gov (United States)

    Veliev, Farida; Briançon-Marjollet, Anne; Bouchiat, Vincent; Delacour, Cécile

    2016-04-01

    Due to its outstanding mechanical and electrical properties as well as chemical inertness, graphene has attracted a growing interest in the field of bioelectric interfacing. Herein, we investigate the suitability of pristine, i.e. without a cell adhesive coating, chemical vapor deposition (CVD) grown monolayer graphene to act as a platform for neuronal growth. We study the development of primary hippocampal neurons grown on bare graphene (transferred on glass coverslip) for up to 5 days and show that pristine graphene significantly improves the neurons adhesion and outgrowth at the early stage of culture (1-2 days in vitro). At the later development stage, neurons grown on coating free graphene (untreated with poly-L-lysine) show remarkably well developed neuritic architecture similar to those cultured on conventional poly-L-lysine coated glass coverslips. This exceptional possibility to bypass the adhesive coating allows a direct electrical contact of graphene to the cells and reveals its great potential for chronic medical implants and tissue engineering. Moreover, regarding the controversial results obtained on the neuronal affinity of pristine graphene and its ability to support neuronal growth without the need of polymer or protein coating, we found that the crystallinity of CVD grown graphene plays an important role in neuronal attachment, outgrowth and axonal specification. In particular, we show that the decreasing crystalline quality of graphene tunes the neuronal affinity from highly adhesive to fully repellent. PMID:26878439

  17. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications

    OpenAIRE

    Ken-ichiro Matsunaga; Michiko Kimoto; Charlotte Hanson; Michael Sanford; Young, Howard A.; Ichiro Hirao

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (K D of 33 pM) survived in human serum at 37 ...

  18. Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic Escherichia coli K88ac+ Fimbriae

    OpenAIRE

    Fang, Lin; Gan, Zhibo; Marquardt, Ronald R.

    2000-01-01

    An affinity chromatography technique was utilized to isolate and purify the receptors of Escherichia coli K88ac+ fimbriae from the mucus of the small intestines of newborn piglets. Purified K88ac+ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B). The immobilized fimbriae were used for the affinity purification of the K88ac+ receptors. Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound recept...

  19. Improved native affinity purification of RNA.

    Science.gov (United States)

    Batey, Robert T; Kieft, Jeffrey S

    2007-08-01

    RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

  20. AFFINITY OF LIGNIN PREPARATIONS TOWARDS GENOTOXIC COMPOUNDS

    Directory of Open Access Journals (Sweden)

    Božena Košíková

    2009-02-01

    Full Text Available The carcinogenicity and mutagenicity of chemicals may be modulated by other chemicals, including those prepared by organic synthesis. Consid-ering the several drawbacks of synthetic compounds vis-a-vis the human organism, the lignin biomass component was examined for this purpose. The binding affinity of lignin samples prepared by chemical and biological modification of lignin products derived from chemical wood treatment towards for N-nitrosodiethylamine (NDA was examined. The protective role of the lignin samples against carcinogenesis was tested on a well-known model carcinogen, N-methyl-N´-nitro-N-nitrosoguanidine (MNNG. The observed ability of a series of lignin preparations to reduce alkylation damage of deoxyribonucleic acid (DNA on hamster cells in vitro could be explained by their affinity to bind N-nitrosoamines. The results indicate that lignin has potential to protect living organisms against damaging effects of different genotoxicants.

  1. Local structure of self-affine sets

    CERN Document Server

    Bandt, Christoph

    2011-01-01

    The structure of a self-similar set with open set condition does not change under magnification. For self-affine sets the situation is completely different. We consider planar self-affine Cantor sets E of the type studied by Bedford, McMullen, Gatzouras and Lalley, for which the projection onto the horizontal axis is an interval. We show that within small square neighborhoods of almost each point x in E, with respect to many product measures on address space, E is well approximated by product sets of an interval and a Cantor set. Even though E is totally disconnected, the limit sets have the product structure with interval fibres, reminiscent to the view of attractors of chaotic differentiable dynamical systems.

  2. Recent Results Regarding Affine Quantum Gravity

    CERN Document Server

    Klauder, John R

    2012-01-01

    Recent progress in the quantization of nonrenormalizable scalar fields has found that a suitable non-classical modification of the ground state wave function leads to a result that eliminates term-by-term divergences that arise in a conventional perturbation analysis. After a brief review of both the scalar field story and the affine quantum gravity program, examination of the procedures used in the latter surprisingly shows an analogous formulation which already implies that affine quantum gravity is not plagued by divergences that arise in a standard perturbation study. Additionally, guided by the projection operator method to deal with quantum constraints, trial reproducing kernels are introduced that satisfy the diffeomorphism constraints. Furthermore, it is argued that the trial reproducing kernels for the diffeomorphism constraints may also satisfy the Hamiltonian constraint as well.

  3. Thermodynamics. Using Affinities to define reversible processes

    CERN Document Server

    Ritacco, Hernán A

    2016-01-01

    In this article a definition of reversible processes in terms of differences in intensive Thermodynamics properties (Affinities) is proposed. This definition makes it possible to both define reversible processes before introducing the concept of entropy and avoid the circularity problem that follows from the Clausius definition of entropy changes. The convenience of this new definition compared to those commonly found in textbooks is demonstrated with examples.

  4. AFFINE TRANSFORMATION IN RANDOM ITERATED FUNCTION SYSTEMS

    Institute of Scientific and Technical Information of China (English)

    熊勇; 史定华

    2001-01-01

    Random iterated function systems (IFSs) is discussed, which is one of the methods for fractal drawing. A certain figure can be reconstructed by a random IFS. One approach is presented to determine a new random IFS, that the figure reconstructed by the new random IFS is the image of the origin figure reconstructed by old IFS under a given affine transformation. Two particular examples are used to show this approach.

  5. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W;

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that (...

  6. A MEMS Dielectric Affinity Glucose Biosensor.

    Science.gov (United States)

    Huang, Xian; Li, Siqi; Davis, Erin; Li, Dachao; Wang, Qian; Lin, Qiao

    2013-06-20

    Continuous glucose monitoring (CGM) sensors based on affinity detection are desirable for long-term and stable glucose management. However, most affinity sensors contain mechanical moving structures and complex design in sensor actuation and signal readout, limiting their reliability in subcutaneously implantable glucose detection. We have previously demonstrated a proof-of-concept dielectric glucose sensor that measured pre-mixed glucose-sensitive polymer solutions at various glucose concentrations. This sensor features simplicity in sensor design, and possesses high specificity and accuracy in glucose detection. However, lack of glucose diffusion passage, this device is unable to fulfill real-time in-vivo monitoring. As a major improvement to this device, we present in this paper a fully implantable MEMS dielectric affinity glucose biosensor that contains a perforated electrode embedded in a suspended diaphragm. This capacitive-based sensor contains no moving parts, and enables glucose diffusion and real-time monitoring. The experimental results indicate that this sensor can detect glucose solutions at physiological concentrations and possesses good reversibility and reliability. This sensor has a time constant to glucose concentration change at approximately 3 min, which is comparable to commercial systems. The sensor has potential applications in fully implantable CGM that require excellent long-term stability and reliability. PMID:24511215

  7. Overview of affinity biosensors in food analysis.

    Science.gov (United States)

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field. PMID:16792079

  8. Overview of affinity biosensors in food analysis.

    Science.gov (United States)

    Patel, Pradip D

    2006-01-01

    The 4 major driving forces that are expected to lead to increased use of affinity biosensors that meet crucial industrial test specifications, e.g., fast, reliable, cost-effective, and use of low-skilled personnel, are (1) strict legislative framework, e.g., recent changes proposed to the European food safety and hygiene legislation, EC No. 178/2002; (2) industrial shift from quality control to quality assurance procedures, e.g., Hazard Analysis Critical Control Point, ensuring effective positioning in the global competitive trade; (3) just-in-time production resulting in 'right' product every time; and (4) consumer demand for safe and wholesome products. The affinity biosensors field has expanded significantly over the past decade, with a projected global biosensors market growth from $6.1 billion in 2004 to $8.2 billion in 2009, representing major industrial sectors (e.g., Pharma, Medicare, and Food). This brief review is targeted to affinity biosensors developed for the food industry and includes research and development leading to biosensors for microbiological and chemical analytes of industrial concern, commercial biosensors products on the market, and examples of future prospects in this diagnostic field.

  9. On constructing purely affine theories with matter

    CERN Document Server

    Cervantes-Cota, Jorge L

    2016-01-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schroedinger's purely affine theory [21], where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  10. On constructing purely affine theories with matter

    Science.gov (United States)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  11. Cellulose binding domain fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Shoseyov, Oded (Karmey Yosef, IL); Shpiegl, Itai (Rehovot, IL); Goldstein, Marc A. (Davis, CA); Doi, Roy H. (Davis, CA)

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  12. Experimental investigation of streamer affinity for dielectric surfaces

    NARCIS (Netherlands)

    Trienekens, D.J.M.; Nijdam, S.; Akkermans, G.; Plompen, I.; Christen, T.; Ebert, U.

    2015-01-01

    We have experimentally investigated the affinity of streamers for dielectric surfaces using stroboscopic imaging and stereo photography. Affinity of streamers for dielectric surfaces was found to depend on a wide set of parameters, including pressure, voltage, dielectric material and di

  13. Quelques remarques sur la notion de modification affine

    OpenAIRE

    Dubouloz, Adrien

    2005-01-01

    in french We construct a global counterpart to the notion of affine modification due to Kaliman and Zaidenberg. This leads to a simple explicit description of the structure of birational affine morphisms between arbitrary quasi-projective varieties.

  14. The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity

    Science.gov (United States)

    Hürlimann, Hans Caspar; Pinson, Benoît; Stadler-Waibel, Martha; Zeeman, Samuel C; Freimoser, Florian M

    2009-01-01

    Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2. PMID:19590579

  15. Fractal Homeomorphism for Bi-affine Iterated Function Systems

    CERN Document Server

    Barnsley, Michael

    2011-01-01

    The paper concerns fractal homeomorphism between the attractors of two bi-affine iterated function systems. After a general discussion of bi-affine functions, conditions are provided under which a bi-affine iterated function system is contractive, thus guaranteeing an attractor. After a general discussion of fractal homeomorphism, fractal homeomorphisms are constructed for a specific type of bi-affine iterated function system.

  16. Biomimetic design of affinity peptide ligand for capsomere of virus-like particle.

    Science.gov (United States)

    Li, Yanying; Liu, Xiaodan; Dong, Xiaoyan; Zhang, Lin; Sun, Yan

    2014-07-22

    Virus-like particle (VLP) of murine polyomavirus (MPV) is a T = 7d icosahedral capsid that self-assembles from 72 capsomeres (Caps), each of which is a pentamer of major coat protein VP1. VLP has great potential in vaccinology, gene therapy, drug delivery, and materials science. However, its application is hindered by high cost downstream processes, leading to an urgent demand of a highly efficient affinity ligand for the separation and purification of Cap by affinity chromatography. Herein a biomimetic design strategy of an affinity peptide ligand of Cap has been developed on the basis of the binding structure of the C-terminus of minor coat protein (VP2-C) on the inner surface of Cap. The molecular interactions between VP2-C and Cap were first examined using all-atom molecular dynamics (MD) simulations coupled with the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method, where V283, P285, D286, W287, L289, and Y296 of VP2-C were identified as the hot spots. An affinity peptide library (DWXLXLXY, X denotes arbitrary amino acids except cysteine) was then constructed for virtual screening sequently by docking with AUTODOCK VINA, binding structure comparison, and final docking with ROSETTA FlexPepDock. Ten peptide candidates were selected and further confirmed by MD simulations and MM/PBSA, where DWDLRLLY was found to have the highest affinity to Cap. In DWDLRLLY, six residues are favorable for the binding, including W2, L4, L6 and Y8 inheriting from VP2-C, and R5 and L7 selected in the virtual screening. This confirms the high efficiency and accuracy of the biomimetic design strategy. DWDLRLLY was then experimentally validated by a one-step purification of Cap from crude cell lysate using affinity chromatography with the octapeptide immobilized on Sepharose gel. The purified Caps were observed to self-assemble into VLP with consistent structure of authentic MPV. PMID:24976378

  17. Duals of Affine Grassmann Codes and Their Relatives

    DEFF Research Database (Denmark)

    Beelen, P.; Ghorpade, S. R.; Hoholdt, T.

    2012-01-01

    Affine Grassmann codes are a variant of generalized Reed-Muller codes and are closely related to Grassmann codes. These codes were introduced in a recent work by Beelen Here, we consider, more generally, affine Grassmann codes of a given level. We explicitly determine the dual of an affine Grassm...

  18. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  19. Crystal structure of the plant dual-affinity nitrate transporter NRT1.1

    Science.gov (United States)

    Sun, Ji; Bankston, John R.; Payandeh, Jian; Hinds, Thomas R.; Zagotta, William N.; Zheng, Ning

    2014-03-01

    Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter that can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr 101 however, how this post-translational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr 101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 dimerizes in the cell membrane and that the phosphomimetic mutation of Thr 101 converts the protein into a monophasic high-affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

  20. Structure-affinity properties of a high-affinity ligand of FKBP12 studied by molecular simulations of a binding intermediate.

    Directory of Open Access Journals (Sweden)

    Lilian Olivieri

    Full Text Available With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant [Formula: see text] of this intermediate should be decreased, resulting in an increase of the affinity constant [Formula: see text]. The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state.

  1. Statistical removal of background signals from high-throughput 1H NMR line-broadening ligand-affinity screens

    International Nuclear Information System (INIS)

    NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional 1H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein 1H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction

  2. Statistical removal of background signals from high-throughput {sup 1}H NMR line-broadening ligand-affinity screens

    Energy Technology Data Exchange (ETDEWEB)

    Worley, Bradley; Sisco, Nicholas J.; Powers, Robert, E-mail: rpowers3@unl.edu [University of Nebraska-Lincoln, Department of Chemistry (United States)

    2015-09-15

    NMR ligand-affinity screens are vital to drug discovery, are routinely used to screen fragment-based libraries, and used to verify chemical leads from high-throughput assays and virtual screens. NMR ligand-affinity screens are also a highly informative first step towards identifying functional epitopes of unknown proteins, as well as elucidating the biochemical functions of protein–ligand interaction at their binding interfaces. While simple one-dimensional {sup 1}H NMR experiments are capable of indicating binding through a change in ligand line shape, they are plagued by broad, ill-defined background signals from protein {sup 1}H resonances. We present an uncomplicated method for subtraction of protein background in high-throughput ligand-based affinity screens, and show that its performance is maximized when phase-scatter correction is applied prior to subtraction.

  3. Analysis of affinely equivalent Boolean functions

    Institute of Scientific and Technical Information of China (English)

    MENG QingShu; ZHANG HuanGuo; YANG Min; WANG ZhangYi

    2007-01-01

    By some basic transforms and invariant theory, we give two results: 1) an algorithm,which can be used to judge if two Boolean functions are affinely equivalent and to obtain the equivalence relationship if they are equivalent. This is useful in studying Boolean functions and in engineering. For example, we classify all 8-variable homogeneous bent functions of degree 3 into two classes; 2) Reed-Muller codes R(4,6)/R(1,6), R(3,7)/R(1,7) are classified efficiently.

  4. On Metrizability of Invariant Affine Connections

    CERN Document Server

    Tanaka, Erico

    2011-01-01

    The metrizability problem for a symmetric affine connection on a manifold, invariant with respect to a group of diffeomorphisms G, is considered. We say that the connection is G-metrizable, if it is expressible as the Levi-Civita connection of a G-invariant metric field. In this paper we analyze the G-metrizability equations for the rotation group G = SO(3), acting canonically on three- and four-dimensional Euclidean spaces. We show that the property of the connection to be SO(3)-invariant allows us to find complete explicit description of all solutions of the SO(3)-metrizability equations.

  5. Latest European coelacanth shows Gondwanan affinities.

    Science.gov (United States)

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  6. Affine Coherent States in Quantum Cosmology

    CERN Document Server

    Malkiewicz, Przemyslaw

    2015-01-01

    A brief summary of the application of coherent states in the examination of quantum dynamics of cosmological models is given. We discuss quantization maps, phase space probability distributions and semiclassical phase spaces. The implementation of coherent states based on the affine group resolves the hardest singularities, renders self-adjoint Hamiltonians without boundary conditions and provides a completely consistent semi-classical description of the involved quantum dynamics. We consider three examples: the closed Friedmann model, the anisotropic Bianchi Type I model and the deep quantum domain of the Bianchi Type IX model.

  7. Measuring an antibody affinity distribution molecule by molecule

    Energy Technology Data Exchange (ETDEWEB)

    Bradbury, Andrew M [Los Alamos National Laboratory; Werner, James H [Los Alamos National Laboratory; Temirov, Jamshid [INVITROGEN

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  8. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    Science.gov (United States)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  9. Connection between the Affine and conformal Affine Toda models and their Hirota's solution

    International Nuclear Information System (INIS)

    It is shown that the Affine Toda models (AT) constitute a gauge fixed version of the Conformal Affine Toda model (CAT). This result enables one to map every solution of the AT models into an infinite number of solutions of the corresponding CAT models, each one associated to a point of the orbit of the conformal group. The Hirota's τ-function are introduced and soliton solutions for the AT and CAT models associated to SL (r+1) and SP (r) are constructed. (author)

  10. Evaluation of Caesalpinia pulcherrima endospermic gum as affinity matrices for galactose-binding lectins interaction

    Directory of Open Access Journals (Sweden)

    Renata Chastinet Braga

    2011-04-01

    Full Text Available Lectins are proteins or glycoproteins able to bind, specifically and reversibly carbohydrates and glycoconjugates. Considering this ability, the utilization of Caesalpinia pulcherrima seeds polysaccharides as an affinity matrix was tested. The endospermic gum were solubilized in distinct concentrations of NaOH and treated with different amounts of epichlorohydrin (ECH forming affinity gels with variable capacity for interaction with galactose- binding lectins. The gel with an ECH/gum ration of 6.0mmol/g was selected as the best affinity matrix. The matrix presented different efficiencies in terms of isolating galactose-binding lectins. C. pulcherrima endospermic galactomannans were purified by ethanol precipitation and the purified galactomannan was crosslinked with the best formulation of gel. The Artocarpus incisa, Ricinus communis and Abrus precatorius lectins showed interactions of 11.5, 17.7 and 47.2mg of retained protein in 1g of gel, respectively; the Artocarpus integrifolia lectin showed the highest affinities (79.7mg/g. The heamaglutination assays confirmed the activity and SDS-PAGE electrophoresis confirmed the isolation of the lectins in a single-step procedure.

  11. Modulating the DNA affinity of Elk-1 with computationally selected mutations.

    Science.gov (United States)

    Park, Sheldon; Boder, Eric T; Saven, Jeffery G

    2005-04-22

    In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

  12. A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

    Science.gov (United States)

    Grecu, Dora; Irudayaraj, Victor Paul Raj; Martinez-Sanz, Juan; Mallet, Jean-Maurice; Assairi, Liliane

    2016-04-01

    The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets. PMID:26923803

  13. The C2 domains of granuphilin are high-affinity sensors for plasma membrane lipids.

    Science.gov (United States)

    Lyakhova, Tatyana A; Knight, Jefferson D

    2014-09-01

    Membrane-targeting proteins are crucial components of many cell signaling pathways, including the secretion of insulin. Granuphilin, also known as synaptotagmin-like protein 4, functions in tethering secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin docks to insulin secretory vesicles through interaction of its N-terminal domain with vesicular Rab proteins; however, the mechanisms of granuphilin plasma membrane targeting and release are less clear. Granuphilin contains two C2 domains, C2A and C2B, that interact with the plasma membrane lipid phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The goal of this study was to determine membrane-binding mechanisms, affinities, and kinetics of both granuphilin C2 domains using fluorescence spectroscopic techniques. Results indicate that both C2A and C2B bind anionic lipids in a Ca(2+)-independent manner. The C2A domain binds liposomes containing a physiological mixture of lipids including 2% PI(4,5)P2 or PI(3,4,5)P3 with high affinity (apparent K(d, PIPx) of 2-5 nM), and binds nonspecifically with moderate affinity to anionic liposomes lacking phosphatidylinositol phosphate (PIPx) lipids. The C2B domain binds with sub-micromolar affinity to liposomes containing PI(4,5)P2 but does not have a measurable affinity for background anionic lipids. Both domains can be competed away from their target lipids by the soluble PIPx analog inositol-(1,2,3,4,5,6)-hexakisphosphate (IP6), which is a positive regulator of insulin secretion. Potential roles of these interactions in the docking and release of granuphilin from the plasma membrane are discussed.

  14. A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

    Science.gov (United States)

    Grecu, Dora; Irudayaraj, Victor Paul Raj; Martinez-Sanz, Juan; Mallet, Jean-Maurice; Assairi, Liliane

    2016-04-01

    The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets.

  15. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    Science.gov (United States)

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  16. A Methodology for Simultaneous Fluorogenic Derivatization and Boronate Affinity Enrichment of 3-Nitrotyrosine Containing Peptides

    Science.gov (United States)

    Dremina, Elena S.; Li, Xiaobao; Galeva, Nadezhda A.; Sharov, Victor S.; Stobaugh, John F.; Schöneich, Christian

    2011-01-01

    We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of non-derivatized peptides, and to enrich them for fluorescent detection and MS identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures. PMID:21855526

  17. A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.

    Science.gov (United States)

    Dremina, Elena S; Li, Xiaobao; Galeva, Nadezhda A; Sharov, Victor S; Stobaugh, John F; Schöneich, Christian

    2011-11-15

    We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures. PMID:21855526

  18. The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

    Science.gov (United States)

    Johnson, Rachel M; Rath, Arianna; Deber, Charles M

    2006-12-01

    Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.

  19. Aspects of affine Toda field theory

    International Nuclear Information System (INIS)

    The report is devoted to properties of the affine Toda field theory, the intention being to highlight a selection of curious properties that should be explicable in terms of the underlying group theory but for which in most cases there are no explanation. The motivation for exploring the ideas contained in this report came principally from the recent work of Zamolodchikov concerning the two dimensional Ising model at critical temperature perturbed by a magnetic field. Hollowood and Mansfield pointed out that since Toda field theory is conformal the perturbation considered by Zamolodchikov might well be best regarded as a perturbation of a Toda field theory. This work made it seem plausible that the theory sought by Zamolodchikov was actually affine E8 Toda field theory. However, this connection required an imaginary value of the coupling constant. Investigations here concerning exact S-matrices use a perturbative approach based on real coupling and the results differ in various ways from those thought to correspond to perturbed conformal field theory. A further motivation is to explore the connection between conformal and perturbed conformal field theories in other contexts using similar ideas. (N.K.)

  20. Exploring Fluorous Affinity by Liquid Chromatography.

    Science.gov (United States)

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  1. Affine Mirkovi\\'c-Vilonen polytopes

    CERN Document Server

    Baumann, Pierre; Tingley, Peter

    2011-01-01

    Each integrable lowest weight representation of a symmetrizable Kac-Moody Lie algebra g has a crystal in the sense of Kashiwara, which describes its combinatorial properties. For a given g, there is a limit crystal, usually denoted by B(-\\infty), which contains all the other crystals. When g is finite dimensional, a convex polytope, called the Mirkovi\\'c-Vilonen polytope, can be associated to each element in B(-\\infty). This polytope sits in the dual space of a Cartan subalgebra of g, and its edges are parallel to the roots of g. In this paper, we generalize this construction to the case where g is a symmetric affine Kac-Moody algebra. The datum of the polytope must however be complemented by partitions attached to the edges parallel to the imaginary root \\delta. We prove that these decorated polytopes are characterized by conditions on their normal fans and on their 2-faces. In addition, we discuss how our polytopes provide an analog of the notion of Lusztig datum for affine Kac-Moody algebras. Our main tool...

  2. Affine conformal vectors in space-time

    Science.gov (United States)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  3. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  4. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  5. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda;

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors...

  6. On-bead chemical synthesis and display of phosphopeptides for affinity pull-down proteomics

    DEFF Research Database (Denmark)

    Malene, Brandt; Madsen, Jens C.; Bunkenborg, Jakob;

    2006-01-01

    We describe a new method for phosphopeptide proteomics based on the solid-phase synthesis of phosphopeptides on beads suitable for affinity pull-down experiments. Peptide sequences containing the Bad Ser112 and Ser136 phosphorylation motifs were used as bait in affinity pull-down experiments...... to determine their ability to bind 14-3-3 proteins. Support-bound peptides were assembled directly on the solid support (PEGA) by standard solid-phase synthesis through a BAL-type handle. The peptides were varied in length and sequence. This synthetic strategy also allowed introduction of a soft electrophile...... (aldehyde) at the C terminus for potential activity-based proteomics. The synthetic support-bound Bad phosphopeptides were able to pull down 14-3-3zeta. Furthermore, Bad phosphopeptides bound endogenous 14-3-3 proteins, and all seven members of the 14-3-3 family were identified by mass spectrometry...

  7. AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

    Science.gov (United States)

    Meyer, Steffen; Woodward, Martin; Hertel, Christina; Vlaicu, Philip; Haque, Yasmin; Kärner, Jaanika; Macagno, Annalisa; Onuoha, Shimobi C; Fishman, Dmytro; Peterson, Hedi; Metsküla, Kaja; Uibo, Raivo; Jäntti, Kirsi; Hokynar, Kati; Wolff, Anette S B; Krohn, Kai; Ranki, Annamari; Peterson, Pärt; Kisand, Kai; Hayday, Adrian

    2016-07-28

    APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility. PMID:27426947

  8. Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody

    DEFF Research Database (Denmark)

    Lassen, K.S.; Bradbury, A.R.; Heegaard, N.H.;

    2008-01-01

    peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide...... (CCK8) in the 1-3 microM range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a K(d1) of 20.1 microM for the monovalent complex. The same...... binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody-antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material...

  9. Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan; Dreyfus, Cyrille; Fleishman, Sarel J.; De Mattos, Cecilia; Myers, Chris A.; Kamisetty, Hetunandan; Blair, Patrick; Wilson, Ian A.; Baker, David (UWASH); (Scripps); (NRL)

    2012-06-19

    We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.

  10. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    Energy Technology Data Exchange (ETDEWEB)

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  11. A Methodology for Simultaneous Fluorogenic Derivatization and Boronate Affinity Enrichment of 3-Nitrotyrosine Containing Peptides

    OpenAIRE

    Dremina, Elena S.; Li, Xiaobao; Galeva, Nadezhda A.; Sharov, Victor S.; Stobaugh, John F.; Schöneich, Christian

    2011-01-01

    We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO2)LER, was employed as a model for method validation. Further, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to p...

  12. Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

    OpenAIRE

    White, Jim F.; Reinhard Grisshammer

    2010-01-01

    BACKGROUND: 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 q...

  13. Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

    International Nuclear Information System (INIS)

    An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 μM concentration of either activated [3H]folate or activated [3H]methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labeling of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms

  14. Metal-conjugated affinity labels: A new concept to create enantioselective artificial metalloenzymes

    KAUST Repository

    Reiner, Thomas

    2013-02-20

    How to train a protein: Metal-conjugated affinity labels were used to selectively position catalytically active metal centers in the binding pocket of proteases. The resulting artificial metalloenzymes achieve up to 82% e.r. in the hydrogenation of ketones. The modular setup enables a rapid generation of artificial metalloenzyme libraries, which can be adapted to a broad range of catalytic conditions. 2013 The Authors.

  15. Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

    OpenAIRE

    White, Jim F.; Grisshammer, Reinhard

    2010-01-01

    Background 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 qu...

  16. Transient interactions between photosynthetic proteins

    NARCIS (Netherlands)

    Hulsker, Rinske

    2008-01-01

    The biological processes that are the basis of all life forms are mediated largely by protein-protein interactions. The protein complexes involved in these interactions can be categorised by their affinity, which results in a range from static to transient complexes. Electron transfer complexes, whi

  17. Affine connection form of Regge calculus

    CERN Document Server

    Khatsymovsky, V M

    2015-01-01

    Regge action is represented analogously to how the Palatini action for general relativity (GR) as some functional of the metric and a general connection as independent variables represents the Einstein-Hilbert action. The piecewise flat (or simplicial) spacetime of Regge calculus is equipped with some world coordinates and some piecewise affine metric which is completely defined by the set of edge lengths and the world coordinates of the vertices. The conjugate variables are the general nondegenerate matrices on the 3-simplices which play a role of a general discrete connection. Our previous result on some representation of the Regge calculus action in terms of the local Euclidean (Minkowsky) frame vectors and orthogonal connection matrices as independent variables is somewhat modified for the considered case of the general linear group GL(4,R) of the connection matrices. As a result, we have some action invariant w. r. t. arbitrary change of coordinates of the vertices (and related GL(4,R) transformations in...

  18. Affine and Projective Tree Metric Theorems

    CERN Document Server

    Harel, Matan; Pachter, Lior

    2011-01-01

    The tree metric theorem provides a combinatorial four point condition that characterizes dissimilarity maps derived from pairwise compatible split systems. A similar (but weaker) four point condition characterizes dissimilarity maps derived from circular split systems (Kalmanson metrics). The tree metric theorem was first discovered in the context of phylogenetics and forms the basis of many tree reconstruction algorithms, whereas Kalmanson metrics were first considered by computer scientists, and are notable in that they are a non-trivial class of metrics for which the traveling salesman problem is tractable. We present a unifying framework for these theorems based on combinatorial structures that are used for graph planarity testing. These are (projective) PC-trees, and their affine analogs, PQ-trees. In the projective case, we generalize a number of concepts from clustering theory, including hierarchies, pyramids, ultrametrics and Robinsonian matrices, and the theorems that relate them. As with tree metric...

  19. Effectively nonlocal metric-affine gravity

    CERN Document Server

    Golovnev, Alexey; Sandstad, Marit

    2015-01-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with non-singular and cosmologically interesting properties may be recovered. Relations between different formulations are analysed at both perturbative and nonperturbative levels taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  20. Effectively nonlocal metric-affine gravity

    Science.gov (United States)

    Golovnev, Alexey; Koivisto, Tomi; Sandstad, Marit

    2016-03-01

    In metric-affine theories of gravity such as the C-theories, the spacetime connection is associated to a metric that is nontrivially related to the physical metric. In this article, such theories are rewritten in terms of a single metric, and it is shown that they can be recast as effectively nonlocal gravity. With some assumptions, known ghost-free theories with nonsingular and cosmologically interesting properties may be recovered. Relations between different formulations are analyzed at both perturbative and nonperturbative levels, taking carefully into account subtleties with boundary conditions in the presence of integral operators in the action, and equivalences between theories related by nonlocal redefinitions of the fields are verified at the level of equations of motion. This suggests a possible geometrical interpretation of nonlocal gravity as an emergent property of non-Riemannian spacetime structure.

  1. Data Stream Clustering With Affinity Propagation

    KAUST Repository

    Zhang, Xiangliang

    2014-07-09

    Data stream clustering provides insights into the underlying patterns of data flows. This paper focuses on selecting the best representatives from clusters of streaming data. There are two main challenges: how to cluster with the best representatives and how to handle the evolving patterns that are important characteristics of streaming data with dynamic distributions. We employ the Affinity Propagation (AP) algorithm presented in 2007 by Frey and Dueck for the first challenge, as it offers good guarantees of clustering optimality for selecting exemplars. The second challenging problem is solved by change detection. The presented StrAP algorithm combines AP with a statistical change point detection test; the clustering model is rebuilt whenever the test detects a change in the underlying data distribution. Besides the validation on two benchmark data sets, the presented algorithm is validated on a real-world application, monitoring the data flow of jobs submitted to the EGEE grid.

  2. Affine trajectory correction for nonholonomic mobile robots

    CERN Document Server

    Pham, Quang-Cuong

    2011-01-01

    Planning trajectories for nonholonomic systems is difficult and computationally expensive. When facing unexpected events, it may therefore be preferable to deform in some way the initially planned trajectory rather than to re-plan entirely a new one. We suggest here a method based on affine transformations to make such deformations. This method is exact and fast: the deformations and the resulting trajectories can be computed algebraically, in one step, and without any trajectory re-integration. To demonstrate the possibilities offered by this new method, we use it to derive position correction, orientation correction, obstacle avoidance and feedback control algorithms for the general class of planar wheeled robots and for a tridimensional underwater vehicle.

  3. Preparation and purification of mouse monoclonal antibody against NMDAR1 by ProteinA - Sepharose CL - 4B affinity chromatography%应用Protein A亲和层析法制备及纯化R1JHL单克隆抗体

    Institute of Scientific and Technical Information of China (English)

    李桂新; 孙长凯; 程桂芝; 罗建红; 李芳; 徐淑君

    2008-01-01

    [目的]制备、纯化N-甲基D-门冬氨酸受体(NMDAR,NR)主亚基RIJHL单克隆抗体.[方法]NR1杂交瘤细胞腹腔注射BalB/C小鼠制备抗体,Protein A-Sepharose CL-4B亲和层析纯化单抗,Western-blot鉴定该单抗特异性.[结果]纯化后单抗识别大鼠脑组织膜蛋白中约115 kD大小的单一条带.[结论]制备、纯化了具有高度特异性的抗NR1单克隆抗体,为进一步研究NMDA受体提供了工具.

  4. Rapid purification of serum IgM from crucian carp,Carassius auratus by Protein A agarose affinity chromatography%用Protein A亲和层析法快速分离纯化鲫血清IgM方法的建立和应用

    Institute of Scientific and Technical Information of China (English)

    吴光辉; 王庆; 巩华; 石存斌; 李华; 吴淑勤

    2010-01-01

    采用Protein A亲和层析法对鲫Carassius auratus血清中的IgM进行快速分离纯化,所得产物用聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western blotting)进行分析检测.结果表明:采用Protein A亲和层析法可以很好地分离到高纯度的鲫血清IgM,电泳条带中重链和轻链清晰可辨,重链、轻链的相对分子质量分别为85 000、25 000左右,无明显杂带;利用纯化的鲫血清IgM免疫小鼠,获得了高效价抗IgM抗血清,可以特异性识别鲫血清和黏液中IgM重链.应用间接ELISA方法对浸泡免疫后的鲫血清和皮肤黏液中抗体的动态进行检测,结果显示:鲫皮肤黏液中的抗体滴度在免疫后第6天达到峰值,血清中抗体滴度在免疫后第15天达到峰值,前者高峰期出现较早,但持续时间短,后者高峰期出现较晚,但持续时间较长.本试验中所建立的Protien A亲和层析法为鱼类抗体制备、病原检测及免疫学相关研究提供了一种便捷的方法.

  5. A Combinatorial Approach to Biophysically Characterise Chemokine-Glycan Binding Affinities for Drug Development

    Directory of Open Access Journals (Sweden)

    Tanja Gerlza

    2014-07-01

    Full Text Available Chemokine binding to glycosaminoglycans (GAGs is recognised to be an important step in inflammation and other pathological disorders like tumor growth and metastasis. Although different ways and strategies to interfere with these interactions are being pursued, no major breakthrough in the development of glycan-targeting drugs has been reported so far. We have engineered CXCL8 towards a dominant-negative form of this chemokine (dnCXCL8 which was shown to be highly active in various inflammatory animal models due to its inability to bind/activate the cognate CXCL8 GPC receptors on neutrophils in combination with its significantly increased GAG-binding affinity [1]. For the development of GAG-targeting chemokine-based biopharmaceuticals, we have established a repertoire of methods which allow the quantification of protein-GAG interactions. Isothermal fluorescence titration (IFT, surface plasmon resonance (SPR, isothermal titration calorimetry (ITC, and a novel ELISA-like competition assay (ELICO have been used to determine Kd and IC50 values for CXCL8 and dnCXCL8 interacting with heparin and heparan sulfate (HS, the proto-typical members of the GAG family. Although the different methods gave different absolute affinities for the four protein-ligand pairs, the relative increase in GAG-binding affinity of dnCXCL8 compared to the wild type chemokine was found by all methods. In combination, these biophysical methods allow to discriminate between unspecific and specific protein-GAG interactions.

  6. Classical affine W-algebras associated to Lie superalgebras

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Uhi Rinn, E-mail: uhrisu1@math.snu.ac.kr [Department of Mathematical Sciences, Seoul National University, GwanAkRo 1, Gwanak-Gu, Seoul 151-747 (Korea, Republic of)

    2016-02-15

    In this paper, we prove classical affine W-algebras associated to Lie superalgebras (W-superalgebras), which can be constructed in two different ways: via affine classical Hamiltonian reductions and via taking quasi-classical limits of quantum affine W-superalgebras. Also, we show that a classical finite W-superalgebra can be obtained by a Zhu algebra of a classical affine W-superalgebra. Using the definition by Hamiltonian reductions, we find free generators of a classical W-superalgebra associated to a minimal nilpotent. Moreover, we compute generators of the classical W-algebra associated to spo(2|3) and its principal nilpotent. In the last part of this paper, we introduce a generalization of classical affine W-superalgebras called classical affine fractional W-superalgebras. We show these have Poisson vertex algebra structures and find generators of a fractional W-superalgebra associated to a minimal nilpotent.

  7. Indefinite Affine Hyperspheres Admitting a Pointwise Symmetry. Part 2

    Directory of Open Access Journals (Sweden)

    Christine Scharlach

    2009-10-01

    Full Text Available An affine hypersurface M is said to admit a pointwise symmetry, if there exists a subgroup G of Aut(T_pM for all p in M, which preserves (pointwise the affine metric h, the difference tensor K and the affine shape operator S. Here, we consider 3-dimensional indefinite affine hyperspheres, i.e. S = HId (and thus S is trivially preserved. In Part 1 we found the possible symmetry groups G and gave for each G a canonical form of K. We started a classification by showing that hyperspheres admitting a pointwise Z_2 × Z_2 resp. R-symmetry are well-known, they have constant sectional curvature and Pick invariant J < 0 resp. J = 0. Here, we continue with affine hyperspheres admitting a pointwise Z_3- or SO(2-symmetry. They turn out to be warped products of affine spheres (Z_3 or quadrics (SO(2 with a curve.

  8. Abacus models for parabolic quotients of affine Weyl groups

    CERN Document Server

    Hanusa, Christopher R H

    2011-01-01

    We introduce abacus diagrams that describe minimal length coset representatives in affine Weyl groups of types B, C, and D. These abacus diagrams use a realization of the affine Weyl group of type C due to Eriksson to generalize a construction of James for the symmetric group. We also describe several combinatorial models for these parabolic quotients that generalize classical results in affine type A related to core partitions.

  9. Proteins at interfaces

    OpenAIRE

    Evers, Florian

    2011-01-01

    Protein adsorption is a fundamental and ubiquitous phenomenon, which has severe implications in the fields of biomaterials as well as bio- and nanotechnology, e.g., in drug delivery, biofouling, the biocompatibility of implants, food chemistry, and biosensors. Therefore, the mechanisms of protein adsorption and controlling the interfacial affinity of proteins have become intriguing and interdisciplinary research topics. In this work, X-ray and neutron reflectometry are the main...

  10. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen;

    2012-01-01

    profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides...... around the phosphorylated residue are important for the binding affinity of ILKAP. We conclude that solid-phase affinity pull-down of proteins from complex mixtures can be applied in phosphoproteomics and systems biology.......Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...

  11. On invariant measures of finite affine type tilings

    OpenAIRE

    Petite, S.

    2004-01-01

    In this paper, we consider tilings of the hyperbolic 2-space, built with a finite number of polygonal tiles, up to affine transformation. To such a tiling T, we associate a space of tilings: the continuous hull Omega(T) on which the affine group acts. This space Omega(T) inherits a solenoid structure whose leaves correspond to the orbits of the affine group. First we prove the finite harmonic measures of this laminated space correspond to finite invariant measures for the affine group action....

  12. A conserved docking site modulates substrate affinity for calcineurin, signaling output, and in vivo function.

    Science.gov (United States)

    Roy, Jagoree; Li, Huiming; Hogan, Patrick G; Cyert, Martha S

    2007-03-23

    Calcineurin, the conserved Ca(2+)/calmodulin-regulated protein phosphatase, mediates diverse aspects of Ca(2+)-dependent signaling. We show that substrates bind calcineurin with varying strengths and examine the impact of this affinity on signaling. We altered the calcineurin-docking site, or PxIxIT motif, in Crz1, the calcineurin-regulated transcription factor in S. cerevisiae, to decrease (Crz1(PVIAVN)) or increase (Crz1(PVIVIT)) its affinity for calcineurin. As a result, the Ca(2+)-dependent dephosphorylation and activation of Crz1(PVIAVN) are decreased, whereas Crz1(PVIVIT) is constitutively dephosphorylated and hyperactive. Surprisingly, the physiological consequences of altering calcineurin-Crz1 affinity depend on the growth conditions. Crz1(PVIVIT) improves yeast growth under several environmental stress conditions but causes a growth defect during alkaline stress, most likely by titrating calcineurin away from other substrates or regulators. Thus, calcineurin-substrate affinity determines the Ca(2+) concentration dependence and output of signaling in vivo as well as the balance between different branches of calcineurin signaling in an overall biological response. PMID:17386265

  13. Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

    Directory of Open Access Journals (Sweden)

    Cicortas Gunnarsson Lavinia

    2009-10-01

    Full Text Available Abstract Background Molecular evolution of carbohydrate binding modules (CBM is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

  14. Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy.

    Science.gov (United States)

    Liu, Zheyi; Wang, Fangjun; Chen, Jin; Zhou, Ye; Zou, Hanfa

    2016-08-26

    Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252.

  15. Modulating the selectivity of affinity absorbents to multi-phosphopeptides by a competitive substitution strategy.

    Science.gov (United States)

    Liu, Zheyi; Wang, Fangjun; Chen, Jin; Zhou, Ye; Zou, Hanfa

    2016-08-26

    Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252. PMID:27470094

  16. Network-of-queues approach to B-cell-receptor affinity discrimination

    Science.gov (United States)

    Felizzi, Federico; Comoglio, Federico

    2012-06-01

    The immune system is one of the most complex signal processing machineries in biology. The adaptive immune system, consisting of B and T lymphocytes, is activated in response to a large spectrum of pathogen antigens. B cells recognize and bind the antigen through B-cell receptors (BCRs) and this is fundamental for B-cell activation. However, the system response is dependent on BCR-antigen affinity values that span several orders of magnitude. Moreover, the ability of the BCR to discriminate between affinities at the high end (e.g., 109M-1-1010M-1) challenges the formulation of a mathematical model able to robustly separate these affinity-dependent responses. Queuing theory enables the analysis of many related processes, such as those resulting from the stochasticity of protein binding and unbinding events. Here we define a network of queues, consisting of BCR early signaling states and transition rates related to the propensity of molecular aggregates to form or disassemble. By considering the family of marginal distributions of BCRs in a given signaling state, we report a significant separation (measured as Jensen-Shannon divergence) that arises from a broad spectrum of antigen affinities.

  17. An affinity pull-down approach to identify the plant cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth

    2013-09-03

    Cyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry - techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants. © Springer Science+Business Media New York 2013.

  18. Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex.

    Science.gov (United States)

    Troeberg, Linda; Mulloy, Barbara; Ghosh, Peter; Lee, Meng-Huee; Murphy, Gillian; Nagase, Hideaki

    2012-04-01

    The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.

  19. The cohomology of the affine Deligne-Lusztig varieties in the affine flag manifold of $GL_2$

    CERN Document Server

    Ivanov, Alexander

    2009-01-01

    This paper studies affine Deligne-Lusztig varieties in the affine flag manifold of GL_2. At first we determine all such varieties up to isomorphy. After this we investigate the representations of the sigma-stabilizer of an element b of the group on the etale cohomology of the affine Deligne-Lusztig variety X_w(b). We describe such representations as inductions from compact subgroups and in terms of noncuspidal representations.

  20. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, T.V.; Holm, A.; Birkelund, S.;

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42) with...