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Natalie J. Saez Hervé Nozach Marilyne Blemont Renaud Vincentelli 《Journal of visualized experiments : JoVE》2014,(89)
Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (www.venomics.eu), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays. 相似文献
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Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments 下载免费PDF全文
Mark Ellis Pareshkumar Patel Marjory Edon Walter Ramage Robert. Dickinson David P. Humphreys 《Biotechnology progress》2017,33(1):212-220
Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017 相似文献
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Mayer M Frey S Koivunen P Myllyharju J Buchner J 《Journal of molecular biology》2004,341(4):1077-1084
Oxidation and folding of secretory proteins in the endoplasmic reticulum (ER) depends on the presence of chaperones and oxidoreductases. Two of the oxidoreductases present in the ER of mammalian cells are protein disulfide isomerase (PDI) and ERp57. In this study, we investigated the influence of ERp57 on the in vitro reoxidation and refolding of an antibody Fab fragment. Our results show that ERp57 shares functional properties with PDI and that both are clearly different from other oxidoreductases. The reactivation of the denatured and reduced Fab fragment was enhanced significantly in the presence of ERp57 with kinetics and redox dependence of the reactivation reaction comparable to those obtained for PDI. These properties were not influenced by the presence of calnexin. Furthermore, whereas PDI cooperates with the immunoglobulin heavy chain binding protein (BiP), no synergistic effect could be observed for BiP and ERp57. These results indicate that the cooperation of the two oxidoreductases with different partner proteins may explain their different roles in the folding of proteins in the ER. 相似文献
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The C-terminal β-hairpin of NS2B (NS2Bc) in the dengue virus NS2B–NS3 protease is required for full enzymatic activity. In crystal structures without inhibitor and in the complex with bovine pancreatic trypsin inhibitor (BPTI), NS2Bc is displaced from the active site. In contrast, nuclear magnetic resonance (NMR) studies in solution only ever showed NS2Bc in the enzymatically active closed conformation. Here we demonstrate by pseudocontact shifts from a lanthanide tag that NS2Bc remains in the closed conformation also in the complex with BPTI. Therefore, the closed conformation is the best template for drug discovery. 相似文献
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Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD 下载免费PDF全文
Rozhkova A Stirnimann CU Frei P Grauschopf U Brunisholz R Grütter MG Capitani G Glockshuber R 《The EMBO journal》2004,23(8):1709-1719
DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways. 相似文献
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Lakshmanane Premkumar Fabian Kurth Simon Neyer Mark A. Schembri Jennifer L. Martin 《The Journal of biological chemistry》2014,289(5):2563-2576
The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (−161 mV) is more reducing than EcDsbC (−130 mV) and EcDsbG (−126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer. 相似文献
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大肠杆菌二硫键形成相关蛋白的结构、功能及其在基因工程表达外源蛋白上的应用 总被引:9,自引:0,他引:9
大肠杆菌分泌蛋白二硫键的形成是一系列蛋白协同作用的结果,主要是Dsb家族蛋白,迄今为止共发现了DsbA、DsbB、DsbC、DsbD、DsbE和DsbG。在体内,DsbA负责氧化两个巯基形成二硫键,DsbB则负责DsbA的再氧化。DsbC和DsbG负责校正DsbA导入的异常二硫键,DsbD则负责对DsbC和DsbG进行再还原,DsbE的功能与DsbD类似。除了直接和二硫键的形成相关外,DsbA、DsbC和DsbG都有分子伴侣功能。它们的分子伴侣功能独立于二硫键形成酶的活性并且对二硫键形成酶活性具有明显的促进作用。基于Dsb蛋白的功能特性,利用它们以大肠杆菌为宿主表达外源蛋白,特别是含有二硫键的蛋白,取得了很多成功的例子。本文简要介绍了这方面的进展,显示Dsb蛋白在促进外源蛋白在大肠杆菌中以可溶形式表达方面具有广阔的应用前景。 相似文献
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Katleen Denoncin Didier Vertommen Isabelle S. Arts Camille V. Goemans Sophie Rahuel-Clermont Joris Messens Jean-Fran?ois Collet 《The Journal of biological chemistry》2014,289(18):12356-12364
We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the l-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing l-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress. 相似文献