共查询到20条相似文献,搜索用时 15 毫秒
1.
Moran D.G. Yoshida S. Fujiyama K. Seki T. Yoshida T. 《World journal of microbiology & biotechnology》1997,13(3):265-267
A truncated Man9-mannosidase gene and a full-length -1,4-galactosyltransferase gene were isolated from human kidney and placenta cDNAs, respectively. Both genes were cloned in plasmid pMAL-c2 to produce fusions with maltose-binding protein. Fusion products were purified by affinity chromatography. Purified enzymes were assayed using pyridyl-amino labelled oligosaccharides as substrates and analysed by HPLC. 相似文献
2.
R Derouiche M Gavioli H Bndetti A Prilipov C Lazdunski R Lloubs 《The EMBO journal》1996,15(23):6408-6415
TolA is an inner membrane protein with three domains: a transmembrane N-terminus and periplasmic central and C-terminal domains. The interaction of TolA with outer membrane porins of Escherichia coli was investigated. Western blot analyses of cell extracts with anti-TolA antibodies indicated that TolA forms high molecular weight complexes specifically with trimeric OmpF, OmpC, PhoE and LamB, but not with OmpA. The interaction of purified TolA domains with purified porins was also studied. TolA interacted with OmpF, PhoE and LamB porins via its central domain, but not with either their denatured monomeric forms or OmpA. Moreover, the presence or absence of lipopolysaccharides associated with trimeric porins did not modify the interactions. These results suggest that the specific interaction of TolA with outer membrane porins might be relevant to the function of Tol proteins. 相似文献
3.
Park JP Lee KB Lee SJ Park TJ Kim MG Chung BH Lee ZW Choi IS Lee SY 《Biotechnology and bioengineering》2005,92(2):160-165
A novel strategy for micropatterning proteins on the surface of polyhydroxyalkanoate (PHA) biopolymer by microcontact printing (microCP) is described. The substrate binding domain (SBD) of the Pseudomonas stutzeri PHA depolymerase was used as a fusion partner for specifically immobilizing proteins on PHA substrate. Enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) fused to the SBD could be specifically immobilized on the micropatterns of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Laser scanning confocal microscopic studies suggested that two fusion proteins were micropatterned in their functionally active forms. Also, antibody binding assay by surface plasmon resonance suggested that protein-protein interaction studies could be carried out using this system. 相似文献
4.
Overexpression of Paenibacillus polymyxa PoxB in Escherichia coli induced the formation of inclusion bodies. An enzyme assay showed that the inclusion bodies exhibited PoxB activity, indicating that they were biologically active. Fusion of GFP and Bacillus subtilis AmyE to the C-terminus of the PoxB also induced the formation of biologically active aggregates when they were overexpressed in E. coli. Therefore, P. polymyxa PoxB can be used as a fusion partner to promote the formation of active inclusion bodies in E. coli. 相似文献
5.
Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines. 相似文献
6.
7.
Mutational analysis of the TolA C-terminal domain of Escherichia coli and genetic evidence for an interaction between TolA and TolB
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The Tol proteins are involved in the outer membrane stability of gram-negative bacteria. The C-terminal domain of TolA was mutagenized to identify residues important for its functions. The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB. 相似文献
8.
Baculoviral polyhedrin as a novel fusion partner for formation of inclusion body in Escherichia coli
Baculoviral polyhedrin, which originated from Autographa californica nuclear polyhedrosis virus (AcNPV), was employed for the first time as a novel fusion partner for expression of foreign proteins in an Escherichia coli system. We characterized the expression of recombinant polyhedrin protein fused to green fluorescent protein (GFP). The polyhedrin fusion protein ( approximately 58 kDa) was successfully expressed as an insoluble inclusion body comprising approximately 30% of the total cellular protein. The E. coli expressing polyhedrin-GFP fusion protein showed higher cell growth ( approximately 1.8-fold) and higher GFP yield ( approximately 3.5-fold) than the strain expressing soluble single GFP. Interestingly, the polyhedrin fusion portion showed almost the same characteristics as the native baculoviral polyhedrin; it was rapidly solubilized under alkaline conditions, similar to the conditions found in the insect midgut. In addition, the polyhedrin fusion portion was rapidly digested by alkaline proteases in insect Plutella xylostella midgut as well as by alpha-chymotrypsin, a protease that has similar properties to insect midgut polyhedra-associated alkaline proteases. These unique properties suggest that baculoviral polyhedrin might be an advantageous fusion partner for production of foreign proteins, especially harmful proteins, in E. coli expression systems. 相似文献
9.
As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli. 相似文献
10.
Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins. 相似文献
11.
Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful. 相似文献
12.
Escherichia coli BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by E. coli. The ompF gene of E. coli BL21(DE3) was first knocked out by using the red operon of bacteriophage lambda to construct E. coli MBEL-BL101. For the excretion of human beta-endorphin as a model protein, the beta-endorphin gene was fused to the C terminus of the E. coli ompF gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-beta-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-beta-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield beta-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of beta-endorphin was obtained. 相似文献
13.
Use of carboxypeptidase Y propeptide as a fusion partner for expression of small polypeptides in Escherichia coli 总被引:1,自引:0,他引:1
The carboxypeptidase Y (CPY) propeptide from Saccharomyces cerevisiae was developed as a fusion partner for the efficient expression of small polypeptides in Escherichia coli. Six consecutive histidine residues (6xHis) were fused to the N-terminus of the CPY propeptide for the facilitated purification of fusion proteins using immobilized metal ion affinity chromatography. In addition, a methionine or the pentapeptide (Asp)(4)-Lys linker was inserted at the junction between the CPY propeptide and the target polypeptide to release the target polypeptide by digestion with cyanogen bromide or enterokinase. Therapeutically valuable peptide hormones, such as salmon calcitonin precursor (sCAL-Gly), a fragment of human parathyroid hormone (hPTH(1-34)), and human glucagon were successfully expressed in E. coli as fusion polypeptides with the fusion partner. SDS-PAGE analyses showed that the majority of the expressed fusion sCAL-Gly and fusion hPTH(1-34) were present in the form of inclusion bodies, whereas about 66% of the expressed human glucagon was in a soluble form. Almost complete cleavage of the fusion polypeptides was obtained by digestion with enterokinase. Reverse-phase HPLC analyses showed that the target polypeptides released from the fusion proteins were identical to their native forms. 相似文献
14.
死亡素与泛素在大肠杆菌中的高效融合表达 总被引:1,自引:0,他引:1
死亡素是由21个氨基酸残基组成的广谱抗菌肽。为了高效表达可溶性的死亡素,本研究利用递归式PCR(recursive PCR, rPCR)扩增了死亡素基因thanatin,并将其和家蝇Musca domestica泛素基因ubiquitin构成嵌合基因,克隆到表达载体pET-32a,再与硫氧还蛋白融合后构建表达载体pET-TRX-UBI-THA。将酶切和测序鉴定正确的质粒转化表达宿主菌BL21,经0.6 mmol/L IPTG诱导,TRX-UBI-THA融合蛋白得到了高效可溶性表达。SDS-PAGE和Western blot检测结果表明融合蛋白的分子量为28.9 kD,与预期的结果一致,表达量占菌体总蛋白的46%。Western blot分析结果显示融合蛋白能与Ni-NTA鏊合物特异性的结合,表明在融合蛋白的N-端带有6×His标签。利用C-端带有6×His标签的泛素C-端水解酶对融合蛋白进行切割,切割产物经Ni2+-NTA亲和柱和HPLC纯化(纯化量为5.4 mg/L),Tricince-SDS-PAGE电泳得到单一的泛素蛋白条带。电喷雾质谱(ESI-MS)分析表明,纯化的泛素分子量为2.57 kD,与通过氨基酸预测的分子量完全一致。利用琼脂孔穴扩散法对泛素活性进行检测,结果显示纯化的泛素对大肠杆菌K12D31和金黄色葡萄球菌Staphylococcus aureus具有较强的活性抑制。本研究表明,利用泛素融合技术可以高效表达可溶性的死亡素。 相似文献
15.
Emmanuelle Bouveret Alain Rigal Claude Lazdunski & Hélène Bénédetti 《Molecular microbiology》1998,27(1):143-157
Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N-terminal domain of colicins is involved in the import process. The N-terminal domains of colicins A and E1 have been shown to interact with TolA, and the N-terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N-terminal domain of all group A colicins, the 'TolA box', was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N-terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N-terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild-type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N-terminal domain interact with TolA and TolB. The colicin A N-terminal domain was also shown to form a trimeric complex with TolA and TolB. 相似文献
16.
Different chimeric proteins combining the non-catalytic C-terminal putative cellulose binding domain of Clostridium cellulovorans endoglucanase-xylanase D (EngD) with its proline-threonine rich region PT-linker, PTCBD(EngD), cellulose binding domain of C. cellulovorans cellulose binding protein A, CBD(CbpA), cohesin domains Cip7, Coh6 and CipC1 from different clostridial species and recombinant antibody binding protein LG were constructed, expressed, purified and analyzed. The solubilities of chimeric proteins containing highly soluble domains Cip7, CipC1 and LG were not affected by fusion with PTCBD(EngD). Insoluble domain Coh6 was solubilized when fused with PTCBD(EngD). In contrast, fusion with CBD(CbpA) resulted in only a slight increase in solubility of Coh6 and even decreased solubility of CipC1 greatly. PTCBD(EngD) and Cip7-PTCBD(EngD) were shown to bind regenerated commercial amorphous cellulose Cuprophan. The purity of Cip7-PTCBD(EngD) eluted from Cuprophan was comparable to that purified by conventional ion exchange chromatography. The results demonstrated that PTCBD(EngD) can serve as a bi-functional fusion tag for solubilization of fusion partners and as a domain for the immobilization, enrichment and purification of molecules or cells on regenerated amorphous cellulose. 相似文献
17.
Improving expression and solubility of rice proteins produced as fusion proteins in Escherichia coli
Tsunoda Y Sakai N Kikuchi K Katoh S Akagi K Miura-Ohnuma J Tashiro Y Murata K Shibuya N Katoh E 《Protein expression and purification》2005,42(2):295-277
For proteins of higher eukaryotes, such as plants, which have large genomes, recombinant protein expression and purification are often difficult. Expression levels tend to be low and the expressed proteins tend to misfold and aggregate. We tested seven different expression vectors in Escherichia coli for rapid subcloning of rice genes and for protein expression and solubility levels. Each expressed gene product has an N-terminal fusion protein and/or tag, and an engineered protease site upstream of the mature rice protein. Several different fusion proteins/tags and protease sites were tested. We found that the fusion proteins and the protease sites have significant and varying effects on expression and solubility levels. The expression vector with the most favorable characteristics is pDEST-trx. The vector, which is a modified version of the commercially available expression vector, pET-32a, contains an N-terminal thioredoxin fusion protein and a hexahistidine tag, and is adapted to the Gateway expression system. However, addition of an engineered protease site could drastically change the expression and solubility properties. We selected 135 genes corresponding to potentially interesting rice proteins, transferred the genes from cDNAs to expression vectors, and engineered in suitable protease sites N-terminal to the mature proteins. Of 135 genes, 131 (97.0%) could be expressed and 72 (53.3%) were soluble when the fusion proteins/tags were present. Thirty-eight mature-length rice proteins and domains (28.1%) are suitable for NMR solution structure studies and/or X-ray crystallography. Our expression systems are useful for the production of soluble plant proteins in E. coli to be used for structural genomics studies. 相似文献
18.
Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners 总被引:2,自引:0,他引:2
Chen YQ Zhang SQ Li BC Qiu W Jiao B Zhang J Diao ZY 《Protein expression and purification》2008,57(2):303-311
It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides. 相似文献
19.
Thierry G.M. Baron Dominique Betemps Martin H. Groschup Jean-Yves Madec 《FEMS immunology and medical microbiology》1999,25(4):379-384
The prion protein (PrP) from sheep was produced in large quantities of entire protein in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence. In contrast, amino-terminal fusion with glutathione S-transferase (GST) revealed a high susceptibility toward cleavage of the protein. Both recombinant proteins were recognised, at variable levels, in Western blots using a panel of antibodies against the 40-56, 89-104, 98-113 and 112-115 sequences of the prion protein, similarly to the abnormal prion protein extracted from scrapie-infected sheep. Interestingly, monoclonal antibody 3F4 was found to react with these three proteins in Western blot. 相似文献
20.
Spector S Flynn JM Tidor B Baker TA Sauer RT 《Protein expression and purification》2003,32(2):317-322
In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal. 相似文献