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1.
Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.  相似文献   

2.
Different parameters that influenced the formation of inclusion bodies in Escherichia coli during production of a fused protein consisting of protein A from Staphylococcus aureus and beta-galactosidase from E. coli were examined. The intracellular expression of the fused protein was controlled by the pR promoter and its temperature-sensitive repressor. The induction temperature, the pH of the cultivation medium, and changes in the amino acid sequence in the linker region between protein A and beta-galactosidase had a profound effect on the formation of inclusion bodies. At 42 degrees C, inclusion bodies were formed only during the first hours after induction, and thereafter all the recombinant protein that was further produced appeared in a soluble and active state. Production at 39 and 44 degrees C resulted in inclusion body formation throughout the production period with 15 to 20% of the produced recombinant protein appearing as inclusion bodies. Cultivating cells without control of pH caused inclusion body formation throughout the induction period, and inclusion body formation increased with decreasing pH, and at least part of the insoluble protein was formed from the pool of soluble fusion protein within the cell. Changes in the amino acid sequence in the linker region between the two parts of the fusion protein abolished inclusion body formation.  相似文献   

3.
The mechanism by which inclusion bodies form is still not well understood, partly because the dynamic processes of the inclusion body formation and its solubilization have hardly been investigated at an individual cell level, and so the important detailed information has not been acquired for the mechanism. In this study, we investigated the in vivo folding and aggregation of Aspergillus phoenicis β-D-galactosidase fused to a red fluorescence protein in individual Escherichia coli cells. The folding status and expression level of the recombinant β-D-galactosidase at an individual cell level was analyzed by flow cytometry in combination with transmission electron microscopy and Western blotting. We found that individual E. coli cells fell into two distinct states, one containing only inclusion bodies accompanied with low galactosidase activity and the other containing the recombinant soluble galactosidase accompanied with high galactosidase activity. The majority of the E. coli cells in the later state possessed no inclusion bodies. The two states of the cells were shifted to a cell state with high enzyme activity by culturing the cells in isopropyl 1-thio-β-D-galactopyranoside-free medium after an initial protein expression induction in isopropyl 1-thio-β-D-galactopyranoside-containing medium. This shift of the cell population status took place without the level change of the β-D-galactosidase protein in individual cells, indicating that the factor(s) besides the crowdedness of the recombinant protein play a major role in the cell state transition. These results shed new light on the mechanism of inclusion body formation and will facilitate the development of new strategies in improving recombinant protein quality.  相似文献   

4.
The protein composition of inclusion bodies produced in recombinant Escherichia coli overproducing Vitreoscilla hemoglobin (VHb) was analyzed by one-dimensional and two-dimensional electrophoresis techniques. Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells. These aggregates also differ in their relative content of VHb and pre-beta-lactamase and are separable by differential centrifugation. Results further suggest that the cytoplasmic protein elongation factor Tu is integrated into VHb inclusion bodies. The presence of the outer membrane proteins OmpA and OmpF in inclusion body preparations is attributed to cell envelope contamination rather than specific involvement in inclusion bodies. The specificity of in vivo protein aggregation is discussed.  相似文献   

5.
6.
The expression of the human IL-2 recombinant gene in E. coli cells was studied. The processes which take place during thermo-induced expression and effect the state of the product were investigated. Experimental data on the membrane localisation of IL-2, the formation of aggregates (inclusion bodies) and polymers were obtained. It was determined that temperature significantly influence the kinetics of the processes of intracellular IL-2 production and IL-2 stability. It is supposed that the cell membrane state plays a determining role in these processes via temperature mediation. Thus, the formation of inclusion bodies described for a number of E. coli recombinant strains is probably stipulated not only by recombinant polypeptides properties, but also by cellular interactions.  相似文献   

7.
The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.  相似文献   

8.
This paper describes the overproduction and purification of the C-terminus polyhistidine-tagged outer membrane protein OprM, which is a part of the MexA-MexB-OprM active efflux system of Pseudomonas aeruginosa. Renaturation of the protein from inclusion bodies of Escherichia coli was achieved using guanidine-HCl as denaturing agent and n-octylpolyoxyethylene (C8POE) and n-octyltetraoxyethylene (C8E4) as nonionic detergents. The refolded protein was purified by ion-exchange and nickel-affinity chromatography. The final yield was 6 mg of pure histidine-tagged OprM per liter of E. coli culture. Renaturation was monitored by the effects of heating prior to SDS-PAGE, using a typical and exclusive property of outer membrane proteins. Immunoblotting revealed that the recombinant protein is addressed to the outer membrane of E. coli, after maturation by excision of its N-terminal signal sequence. Complementation of an oprM deletion mutant with the plasmid encoded histidine-tagged OprM protein restored antibiotic susceptibilities to wild-type levels, demonstrating functionality of recombinant OprM.  相似文献   

9.
Mallory bodies represent cytokeratin-rich inclusion bodies which occur characteristically but not exclusively in human alcoholic liver disease and experimentally in mice during chronic intoxication with drugs. We report the first in vitro cell system of Mallory body induction. In clone 9 rat hepatocytes stably transfected to express an ER-retained T126M-aquaporin-2 (AQP2), on the mean 40% of the cells contained cytokeratin-rich inclusion bodies. By electron microscopy, their structure corresponded to that of genuine Mallory bodies. Such inclusion bodies were not detectable in clone 9 rat hepatocytes stably expressing a Golgi apparatus/lysosome-retained E258K-aquaporin-2. Proteasome inhibition increased the number of Mallory body-containing T126M-AQP2-expressing clone 9 hepatocytes to 60% on average. Proteasome inhibition in non-transfected, cytokeratin meshwork-forming clone 9 hepatocytes resulted in Mallory body formation on average in 6% of cells. Collectively, these data suggest that in the described in vitro cell system, Mallory body formation is induced by the presence of non-native protein conformers and point to the involvement of the proteasomal digestive system. The here reported in vitro system will be useful in studies about the biogenesis and progression of Mallory bodies, their relationship to aggresomes, and the role of inclusion bodies in the pathogenesis of cell damage.  相似文献   

10.
The Erm family of methyltransferases confers the MLS antibiotic resistance to pathogenic microorganism through the mono- or dimethylation of a single adenine residue in 23S rRNA, which is known as the target site for modification. One of the erm genes, ermSF was cloned from Streptomyces fradiae NRRL 2702 by PCR and overexpressed in E. coli BL21(DE3) as both a soluble protein and insoluble aggregate (inclusion body) using the T7 promoter driven expression vector, pET23b. Even though most of the overexpressed protein existed as an inclusion body, E. coli cells showed resistance to erythromycin. The lowering of incubation temperature from 37 degrees C to 22 degrees C facilitated the purification of the protein by increasing the fraction of soluble protein. The soluble protein was purified using immobilized metal ion (Ni2+) affinity chromatography in a one-step manner to the apparent homogeneity. The 23S rRNA of E. coli was found to be a good substrate for the purified ErmSF.  相似文献   

11.
UL16-binding proteins (ULBPs) are markers of cellular stress which are upregulated on the surface of virus-infected and tumor cells. Recognition of ULBP1 by the activating receptor NKG2D on the surface of cytotoxic natural killer (NK) and T cells promotes lysis of cells expressing ULBP1 and is an important mechanism of immune surveillance. We report a robust method for the generation of large quantities of crystal-grade recombinant ULBP1 protein. The extracellular portion of human ULBP1 was cloned into a T7 expression vector for expression in Escherichia coli. Unpaired cysteines in the sequence which are predicted not to be involved in the intramolecular disulfide bond formation were mutated to serine. ULBP1 was expressed in E. coli BL21 (DE3) pLysS cells as inclusion bodies. Purified inclusion bodies were solubilized by denaturation in guanidine, and refolded by slow dilution. The refolded protein was purified by size exclusion gel filtration and anion exchange chromatography. Furthermore, we have identified conditions optimal for the crystallization of this protein and have obtained initial diffraction data to 4.6? from these crystals.  相似文献   

12.
Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coli.  相似文献   

13.
Medwid RD  Krebs L  Welch S 《BioTechniques》2007,43(6):777-782
Many types of commercially valuable recombinant proteins produced by fermentation are expressed at high levels in Escherichia coli. Often, high-level expression in the host results in the formation of insoluble inclusion bodies. The release of these intracellular inclusion bodies from E. coli following cell disruption is a requirement for further downstream recovery. The ability to discern between intact unruptured cells and granules released from broken cells can provide valuable information for improving recovery yields in downstream purification. This paper describes a rapid and sensitive cytometry-based method that allows the simultaneous measurement of intact heat-killed E. coli and inclusion bodies using staining with nucleic acid binding fluorochromes.  相似文献   

14.
The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases.  相似文献   

15.
大肠杆菌是表达重组蛋白最常用的宿主之一。利用大肠杆菌分泌途径胞外表达重组蛋白具有可促进蛋白正确折叠,有效减少包涵体形成,简化纯化工序等诸多优势,近年来备受关注。其中,大肠杆菌I型分泌途径具有分泌表达速度快,蛋白活性高,对宿主代谢无影响等特点,是目前应用最广泛的分泌途径之一。综述了大肠杆菌I型分泌系统的元件组成和分泌机理及提高I型分泌系统蛋白表达量的有效策略,为重组蛋白生产应用提供了理论依据。  相似文献   

16.
DNA microarrays revealed that expression of ycfR, which encodes a putative outer membrane protein, is significantly induced in Escherichia coli biofilms and is also induced by several stress conditions. We show that deletion of ycfR increased biofilm formation fivefold in the presence of glucose; the glucose effect was corroborated by showing binding of the cyclic AMP receptor protein to the ycfR promoter. It appears that YcfR is a multiple stress resistance protein, since deleting ycfR also rendered the cell more sensitive to acid, heat treatment, hydrogen peroxide, and cadmium. Increased biofilm formation through YcfR due to stress appears to be the result of decreasing indole synthesis, since a mutation in the tnaA gene encoding tryptophanase prevented enhanced biofilm formation upon stress and adding indole prevented enhanced biofilm formation upon stress. Deleting ycfR also affected outer membrane proteins and converted the cell from hydrophilic to hydrophobic, as well as increased cell aggregation fourfold. YcfR seems to be involved in the regulation of E. coli K-12 biofilm formation by decreasing cell aggregation and cell surface adhesion, by influencing the concentration of signal molecules, and by interfering with stress responses. Based on our findings, we propose that this locus be named bhsA, for influencing biofilm through hydrophobicity and stress response.  相似文献   

17.
Recent evidence suggests that proliferating cells polarize damaged proteins during mitosis to protect one cell from aging, and that the structural conformation of damaged proteins mediates their toxicity. We report that the growth, resistance to stress, and differentiation characteristics of a cancer cell line (PC12) with an inducible Huntingtin (Htt) fused to enhanced green fluorescent protein (GFP) are dependent on the conformation of Htt. Cell progeny containing inclusion bodies have a longer cell cycle and increased resistance to stress than those with diffuse Htt. Using live imaging, we demonstrate that asymmetric division resulting from a cell containing a single inclusion body produces sister cells with different fates. The cell that receives the inclusion body has decreased proliferation and increased differentiation compared with its sister cell without Htt. This is the first report that reveals a functional consequence of the asymmetric division of damaged proteins in mammalian cells, and we suggest that this is a result of inclusion body-induced proteasome impairment.  相似文献   

18.
Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.  相似文献   

19.
Culture conditions affecting the formation of beta-galactosidase inclusion bodies in E. coli were examined. High temperature, early induction, high salt concentration and low aeration were all found to favour an increase of insoluble beta-galactosidase and the formation of visible inclusion bodies. The ratio of soluble to insoluble beta-galactosidase decreased during the course of cell growth. When assayed for beta-galactosidase activity, the inclusion bodies were enzymatically active with a specific activity of one third that of soluble beta-galactosidase. The activity remained associated with the inclusion bodies on washing with detergent and high ionic strength buffers. These results suggest that inclusion bodies can contain correctly folded protein.  相似文献   

20.
猪α1-干扰素的基因改造与高效原核表达   总被引:15,自引:0,他引:15  
poIFNα1基因中含有大量的大肠杆菌稀有密码子,为了获得高表达,使用了大肠杆菌的偏爱密码子,人工合成了poIFN|α1成熟蛋白编码基因。在保留编码蛋白序列的同时,使其5′端A+T的含量增加到最大限度,并将其终止密码子改为TAA。将合成的poIFNα1成熟蛋白编码基因插入原核单纯表达载体pRLC中,转化大肠杆菌DH5α。实现了poIFNα1在大肠杆菌中的高效表达,表达产物以包涵体形式存在。纯化的包涵体经含DTT的6 mol/L盐酸胍的变性液溶解及含GSHGSSG的复性液复性处理,复性后的表达产物浓缩后经凝胶层析纯化,细胞病变抑制法测定表明,重组poIFNα1具有较高的抗病毒活性,约为6.4x106u/mg。   相似文献   

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