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1.
2.
Escherichia. coli cells expressing porcine growth hormone were grown in a batch fermentation process. The expression level was estimated to be nearly 40% of the total cellular protein after 2–3 h of induction with 1?mM isopropyl β-d-thiogalactoside. Porcine growth hormone expressed as inclusion bodies was solubilized in 8 M urea. Refolding conditions following a dilution protocol in the presence of β-mercaptoethanol or using a glutathione pair were tested. Reverse phase-HPLC was applied to distinguish oxidized, misfolded and reduced forms of the hormone. A ratio of reduced to oxidized glutathione equal to 2/1 was chosen to avoid the formation of misfolded forms at high protein concentration.  相似文献   

3.
The South-Paris Yeast Structural Genomics Project aims at systematically expressing, purifying and determining the structure of S. cerevisiae proteins with no detectable homology to proteins of known structure. We brought 250 yeast ORFs to expression in E. coli, but 37% of them form inclusion bodies. This important fraction of proteins that are well expressed but lost for structural studies prompted us to test methodologies to recover these proteins. Three different strategies were explored in parallel on a set of 20 proteins: (1) refolding from solubilized inclusion bodies using an original and fast 96-well plates screening test, (2) co-expression of the targets in E. coli with DnaK-DnaJ-GrpE and GroEL-GroES chaperones, and (3) use of the cell-free expression system. Most of the tested proteins (17/20) could be resolubilized at least by one approach, but the subsequent purification proved to be difficult for most of them.  相似文献   

4.
肿瘤坏死因子相关凋亡诱导配体(Ap02L/TRAIL)是一种新型的抗肿瘤蛋白。但在大肠杆菌表达系统中,表达的TRAIL蛋白往往容易聚集形成包涵体。本文对重组TRAIL包涵体在胞内重折叠转化成可溶性TRAIL蛋白作了初步探索,结果表明:当蛋白表达后,迅速降低温度至30℃并添加50μg/mL氯霉素继续培养4h,可溶性TRAIL产量可提高至16.7%。这一结果在3.7L罐上也得到了证实,可溶性TRAIL表达量达到14.5%。  相似文献   

5.
Cho TH  Ahn SJ  Lee EK 《Bioseparation》2001,10(4-5):189-196
To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we proposed a solid-phase refolding method integrated with the expanded bed adsorption chromatography. The model protein was a fusion protein of recombinant human growth hormone and a glutathione S-transferase fragment. It was demonstrated that the inclusion body proteins in the cell homogenate could be directly refolded with higher yield. To verify the applicability of this method, we have tested with success three types of the starting materials, i.e., rhGH monomer, inclusion bodies containing the fusion protein, and the E. coli cell homogenate. This direct refolding process could reduce the number of the renaturation steps required and allow the refolding at a higher concentration, approximately 2 mg fusion protein per ml resin. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

6.
Shen YL  Xia XX  Zhang Y  Liu JW  Wei DZ  Yang SL 《Biotechnology letters》2003,25(24):2097-2101
As a new member of tumor necrosis factor (TNF) superfamily, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) was produced mainly as inclusion bodies by recombinant Escherichia coli with a temperature-inducible expression system. High concentrations of both biomass (65 g dry cells l(-1)) and inactive TRAIL (4.8 g l(-1)) were obtained by applying a high-cell-density cultivation procedure. After the inclusion bodies were washed and solubilized. TRAIL refolded when at 1 mg ml(-1) by a simple pulse dilution method with a 35% yield. Renatured TRAIL was purified to electrophoretic homogeneity by one-step immobilized metal affinity chromatography. The purified TRAIL showed strong cytotoxicity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg ml(-1).  相似文献   

7.
Substantial progress has been made towards understanding the folding mechanisms of proteins in vitro and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible clues for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the past decade are summarized.  相似文献   

8.
Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble pro- teins following dilution refoiding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical proper- ties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high effi- cacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight (R2= 0.98, P = 0.000, F-test). Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refold- ing developed here could be used to predict the refold- ing yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refoiding from IBs according to their intrinsic properties.  相似文献   

9.
Aims: To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. Methods and Results: The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca2+ ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg−1 protein; it also revealed broad pH and temperature ranges of 5·0 to 8·0 and 25 to 70°C, respectively. The Km value of phytase for hydrolysis of sodium phytate has been determined as 0·392 mmol l−1. The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95°C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26·81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1·978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a β-propeller structure of phytase. Conclusions: This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. Significance and Impact of the Study: Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity.  相似文献   

10.
Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.  相似文献   

11.
We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010  相似文献   

12.
包含体蛋白质的复性研究进展   总被引:20,自引:0,他引:20  
包含体的形成是异源蛋白质在大肠杆菌中高效表达的必然结果,也是目前产生重组蛋白质最有效的方法之一。不可溶、无生物活性的包含体必须经过变性、复性才能获得天然结构,完整特定的生物学功能。聚集是造成重组蛋白质复性产率低下的主要因素,因此理解蛋白质聚集机制,减少和防止聚集的发生是建立高效、高产率复性方法的关键。分子伴侣、低分子量添加物等在复性过程中的应用及新的复性方法的建立都大大提高了重组蛋白质复性产率。  相似文献   

13.
重组蛋白复性技术研究进展   总被引:6,自引:0,他引:6  
本对近年来重组蛋白复性技术的研究进行了评述。比较分析了液相和固相复性的各种方法,提出了复性优化的方案,介绍了在化学复性基础上发展物理复性如高压复性法的新思路。  相似文献   

14.
描述了大肠杆菌异源重组蛋白质的形成、制备、变性和复性,综述了国内外变性、复性的有效方法。  相似文献   

15.
Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies, In case of expression of eukaryotic proteins containing cysteine, which may form disulfide bonds in the native active protein, often nonnative inter- and intramolecular disulfide bonds exist in the inclusion bodies. Hence, several methods have been developed to isolate recombinant eukaryotic polypeptides from inclusion bodies, and to generate native disulfide bonds, to get active proteins. This article summarizes the different steps and methods of isolation and renaturation of eukaryotic proteins containing disulfide bonds, which have been expressed in E. coli as inclusion bodies, and shows which methods originally developed for studying the folding mechanism of naturally occurring proteins have been successfully adapted for reactivation of recombinant eukaryotic proteins. (c) 1993 John Wiley & Sons, Inc.  相似文献   

16.
Protein folding and chaperonins   总被引:1,自引:0,他引:1  
The folding of polypeptide chains in cells, following either translation or translocation through membranes, must take place under conditions of extremely high protein concentrations. In addition, folding into a correct structure must occur in the presence of other rapidly folding species, and at temperatures known to destabilize aggregation-prone folding intermediates. To facilitate folding in vivo, molecular chaperones have evolved that stabilize protein folding intermediates, thus partitioning them towards a pathway leading to the native state rather than forming inactive aggregated structures.  相似文献   

17.
Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.  相似文献   

18.
The human TRAIL gene (encoding residues 114-281) was synthesized by PCR and cloned into plasmid pET-32a. High level expression (1.5 g l(-1)) of thioredoxin/TRAIL fusion was achieved in Escherichia coli strain BL21(DE3), mainly as inclusion bodies. Refolded fusion thioredoxin/TRAIL was cleaved by enteropeptidase and TRAIL was separated from thioredoxin on Ni-NTA agarose. High yield (400 mg l(-1)) of TRAIL without N-terminal methionine and His tag was obtained. Sedimentation coefficient demonstrated that 98% of TRAIL formed trimers. TRAIL formed crystals of space group P3 (1) with unit-cell dimensions a = b = 72.5 A, c = 141.5 A. Apoptosis induced in HeLa cells by purified TRAIL was 5-fold enhanced by emetine.  相似文献   

19.
A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.  相似文献   

20.
Heterologous overexpression of genes in Escherichia coli has made it possible to obtain high titers of recombinant proteins. However, this can result in the formation of aggregated protein particles known as ‘inclusion bodies’. Protein sequestered as inclusion body is inactive and needs to be converted back to its functional form by refolding using appropriate techniques. In the current study inclusion bodies of the enzyme aminoglycoside nucleotidyl transferase (or ANT(2″)‐Ia) were first solubilized in urea and subsequently subjected to thermal cycling under controlled conditions as part of the refolding strategy. Thermal cycling led to disaggregation of the individual protein chains and simultaneously refolding the released protein molecules to their native state. The optimum condition was identified as 10–80°C thermal cycling at 3°C s?1 for 2 h. Enzyme activity measurements showed that thermal cycling under optimized conditions resulted in 257% activity recovery when compared with nonrefolded protein. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:133–139, 2017  相似文献   

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