共查询到20条相似文献,搜索用时 31 毫秒
1.
Sushil Prasad Sati Saurabh Kumar Singh Nirbhay Kumar Amit Sharma 《European journal of biochemistry》2002,269(21):5259-5263
The presence of extra N- and C- terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over-expressed using the pMAL-p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N-terminal or smaller C-terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over-expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N-terminus and a C-terminal 'solubilization tag'. This system may allow large-scale production of those proteins that have a tendency to misfold during expression. 相似文献
2.
Sørensen HP Sperling-Petersen HU Mortensen KK 《Protein expression and purification》2003,32(2):252-259
Recombinant streptavidin is extremely difficult to express at high levels in the cytoplasm of Escherichia coli without the formation of inclusion bodies. Fusing a solubility enhancing partner to an aggregation prone protein is a widely used tool to circumvent inclusion body formation. Here, we use streptavidin as a target protein to test the properties of N-terminal fragments of translation initiation factor IF2 from E. coli as a solubility partner. Domain I (residue 1-158) of IF2 is superior to the well-established solubility partners maltose-binding protein (MBP) and NusA for soluble expression of active streptavidin. The number of active streptavidin molecules isolated by chromatography is increased threefold when domain I is used as solubility partner as compared to MBP or NusA. The relatively small size, high expressivity, and extreme solubility make domain I of IF2 an ideal partner for streptavidin and may also prevent other recombinant proteins such as ScFv antibodies from being expressed as insoluble aggregates in the cytoplasm of E. coli. 相似文献
3.
The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins. 相似文献
4.
Annette Haacke Gabriele Fendrich Paul Ramage Martin Geiser 《Protein expression and purification》2009,64(2):185-193
The recombinant expression of eukaryotic proteins in Escherichia coli often results in protein aggregation. Several articles report on improved solubility and increased purification yields of individual proteins upon over-expression of E. coli chaperones but this effect might potentially be protein-specific. To find out whether chaperone over-expression is a generally applicable strategy for the production of human protein kinases in E. coli, we analyzed 10 kinases, mainly as catalytic domain constructs. The kinases studied, namely c-Src, c-Abl, Hck, Lck, Igf1R, InsR, KDR, c-Met, b-Raf and Irak4, belong to the tyrosine and tyrosine kinase-like groups of kinases. Upon over-expression of the E. coli chaperones DnaK/DnaJ/GrpE and GroEL/GroES, the yields of 7 from 10 polyhistidine-tagged kinases were increased up to 5-fold after nickel-affinity purification (IMAC). Additive over-expression of the chaperones ClpB and/or trigger factor showed no further improvement. Co-purification of DnaJ and GroEL indicated incomplete kinase folding, therefore, the oligomerization state of the kinases was determined by size-exclusion chromatography. In our study, kinases behave in three different ways. Kinases where yields are not affected by E. coli chaperone over-expression e.g. c-Src elute in the monomeric fraction (category I). Although IMAC yields increase upon chaperone over-expression, InsR and b-Raf kinase are present as soluble aggregates (category II). Igf1R and c-Met kinase catalytic domains are partially complexed with E. coli chaperones upon over-expression; however, they show 2-fold increased yields of monomer (category III). Together, our results suggest that the benefits of chaperone over-expression on the production of protein kinases in E. coli are indeed case-specific. 相似文献
5.
Kim CW Han KS Ryu KS Kim BH Kim KH Choi SI Seong BL 《Protein science : a publication of the Protein Society》2007,16(4):635-643
The fusion of soluble partner to the N terminus of aggregation-prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol. The chaperone-like functions of the upstream fusion partner in the artificial multidomain proteins could occur in de novo folding of native multidomain proteins. Here, we show that the N-terminal domains of three E. coli multidomain proteins such as lysyl-tRNA synthetase, threonyl-tRNA synthetase, and aconitase are potent solubility enhancers for various C-terminal heterologous proteins. The results suggest that the N-terminal domains could act as solubility enhancers for the folding of their authentic C-terminal domains in vivo. Tandem repeat of N-terminal domain or insertion of aspartic residues at the C terminus of the N-terminal domain also increased the solubility of fusion proteins, suggesting that the solubilizing ability correlates with the size and charge of N-terminal domains. The solubilizing ability of N-terminal domains would contribute to the autonomous folding of multidomain proteins in vivo, and based on these results, we propose a model of how N-terminal domains solubilize their downstream domains. 相似文献
6.
Pons JI Rodríguez S Madrid C Juárez A Nieto JM 《Protein expression and purification》2004,35(2):293-297
Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification. This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E. coli. Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins. Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other. We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E. coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies). We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies. Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E. coli. 相似文献
7.
将人源肿瘤坏死因子Ⅰ型受体(hTNFR1)基因克隆到pET-22b表达载体,成功构建了重组表达质粒pETH1,电转到Escherichia coli BL21(DE3)表达菌株中进行摇瓶发酵。实现了hTNFR1在大肠杆菌表达系统中的重组表达。但目的蛋白全部以包涵体的形式存在于沉淀中。为了提高hTNFR1在大肠杆菌中的可溶性表达,融合标签和分子伴侣两种策略被实施用于辅助hTNFR1的可溶性表达。结果表明,在hTNFR1的N端融合NusA标签后,hTNFR1的可溶性有一定提高;在NusA-hTNFR1基础上,过表达了7种分子伴侣,筛选出tig分子伴侣对hTNFR1蛋白可溶性表达有明显的促进作用,可溶性表达量约占总量的90%;对优化后的hTNFR1表达系统的可溶性蛋白进行Ni-NTA亲和层析纯化后,TEV蛋白酶酶切去除N端的NusA标签,结合Western blot分析鉴定,获得了大量高纯度的hTNFR1蛋白。研究结果为进一步研究hTNFR1的生理学活性及其在疾病治疗方面的应用奠定了良好基础。 相似文献
8.
To better investigate the relationship between sequence, stability, and folding, the Streptococcus pyogenes collagenous domain CL (Gly-Xaa-Yaa)(79) was divided to create three recombinant triple helix subdomains A, B, and C of almost equal size with distinctive amino acid features: an A domain high in polar residues, a B domain containing the highest concentration of Pro residues, and a very highly charged C domain. Each segment was expressed as a monomer, a linear dimer, and a linear trimer fused with the trimerization domain (V domain) in Escherichia coli. All recombinant proteins studied formed stable triple helical structures, but the stability varied depending on the amino acid sequence in the A, B, and C segments and increased as the triple helix got longer. V-AAA was found to melt at a much lower temperature (31.0 °C) than V-ABC (V-CL), whereas V-BBB melted at almost the same temperature (~36-37 °C). When heat-denatured, the V domain enhanced refolding for all of the constructs; however, the folding rate was affected by their amino acid sequences and became reduced for longer constructs. The folding rates of all the other constructs were lower than that of the natural V-ABC protein. Amino acid substitution mutations at all Pro residues in the C fragment dramatically decreased stability but increased the folding rate. These results indicate that the thermostability of the bacterial collagen is dominated by the most stable domain in the same manner as found with eukaryotic collagens. 相似文献
9.
Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies. 相似文献
10.
Suhre MH Wegele H Wandinger SK 《International journal of biological macromolecules》2006,39(1-3):23-28
Here we report the recombinant expression of the catalytically active phosphatase domain of the Saccharomyces cerevisiae protein phosphatase 1 (Ppt1) in E. coli. Ppt1 consists of two domains: a 20 kDa TPR (tetratricopeptide repeat) domain, which mediates protein-protein interactions and directs Ppt1 to potential substrate proteins, e.g. the molecular chaperone Hsp90. The second, a 40 kDa phosphatase domain, exhibits catalytic activity and dephosphorylates phosphorylated serine/threonine residues of respective substrate proteins. The Ppt1 phosphatase domain was cloned and expressed in E. coli in unsoluble inclusion bodies. After isolating these, the aggregates were denatured with guanidinium hydrochloride and soluble protein was purified using affinity chromatography. Optimal renaturation conditions led to large amounts of the refolded phosphatase domain in high purity. Interestingly, further enzymatic studies revealed that the domain is not only correctly folded, but also shows higher catalytic activity compared to the full length protein. 相似文献
11.
Sardana V Xu B Zugay-Murphy J Chen Z Sardana M Darke PL Munshi S Kuo LC 《Protein expression and purification》2004,34(2):190-196
Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity. Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-terminus transmembrane domain. Although each of the proteins expresses in inclusion bodies, a generalized protocol has been developed to solubilize, refold, and purify these BACE1 variants. The resultant proteins are homogeneous and monodispersed in solution. Each possesses a unique N-terminus. Activity assays using the peptide substrate 7-methoxycoumarin-4-yl-SEVNLDAEFK-2,4-dinitrophenyl-RR, corresponding to the beta-secretase cleavage sequence in the amyloid precursor protein with the Swedish mutations of N(670)L(671) substituting for the residues K(670)M(671), reveal a kcat and KM of 9.3 min(-1) and 55 microM, respectively. 相似文献
12.
FtsY is a signal recognition particle receptor in Escherichia coli that mediates the targeting of integral membrane proteins to translocons by interacting with both signal recognition particle (SRP)-nascent polypeptide-ribosome complexes and the cytoplasmic membrane. Genes encoding the N-terminal segments of Streptomyces lividans FtsY were fused to a gene encoding the E. coli FtsY NG domain (truncated versions of FtsY lacking the transient membrane-anchor domain at the N-terminus), introduced into a conditional ftsY-deletion mutant of E. coli, and expressed in trans to produce chimeric FtsY proteins. Under FtsY-depleted conditions, strains producing chimeric proteins including 34 N-terminal hydrophobic residues grew whereas strains producing chimeric proteins without these 34 residues did not. A strain producing the chimeric protein comprising the 34 residues and NG domain processed beta-lactamase, suggesting that the SRP-dependent membrane integration of leader peptidase was restored in this strain. These results suggest that the N-terminal hydrophobic segment of FtsY in this Gram-positive bacterium is responsible for its interaction with the cytoplasmic membrane. 相似文献
13.
Proteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and IbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli. 相似文献
14.
15.
Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli. 相似文献
16.
17.
Chen J Acton TB Basu SK Montelione GT Inouye M 《Journal of molecular microbiology and biotechnology》2002,4(6):519-524
Protein misfolding resulting in the formation of inclusion bodies is one of the major problems during protein overexpression in Escherichia coil. In this paper, we introduce a new method, which is simply to heat shock a cell culture prior to protein induction, allowing effective enhancement of the solubility and thereby the yield of overexpressed proteins in E. coli. Using this method, we show that the solubility of the E. coli protein KsgA-AN is significantly increased when overexpressed from a T7 promoter. In addition, we also show that the solubility of several Caenorhabditis elegans proteins are also enhanced after heat-shock treatment when expressed in E. coli. Taken together, these results suggest that the "heat-shock protocol" is a generalizable and useful method for increasing the solubility of many proteins overexpressed in E. coli. 相似文献
18.
人EGF受体L2结构域在大肠杆菌中表达、包涵体柱上复性及纯化 总被引:3,自引:0,他引:3
以PCR方法从克隆的EGFR胞外区cDNA中扩增编码EGFR-L2结构域的DNA片段,在其3′端加入编码His6标签的序列,与pET-3c连接构建EGFR-L2原核表达载体。该蛋白在大肠杆菌BL21(DE3)中获得高效表达,免疫印迹分析表明表达产物全部以包涵体形式存在,分步透析法和稀释法都不能获得可溶性复性产物,而Ni2+-NTA柱上复性法不仅能够获得可溶性的EGFR-L2蛋白,而且产物同时得到高度纯化,纯度>95%,复性的EGFR-L2与其配基EGF具有特异性的结合活性,但亲和力较低。这表明His6标签不但便于纯化目标蛋白,而且可利用Ni2+-NTA柱进行柱上复性,适用于不易通过常规方法复性的重组蛋白的制备。 相似文献
19.
Vertiev IuV Zdanovskiĭ AG Borinskaia SA Martin T Gening EL Iankovskiĭ NK 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》2000,(4):3-7
Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin. 相似文献
20.
Many recombinant proteins overexpressed in Escherichia coli are generally misfolded, which then aggregate and accumulate as inclusion bodies. The catalytic domain (CD) of bovine and human beta1,4-galactosyltransferase (beta4Gal-T), expressed in E. coli, it also accumulates as inclusion bodies. We studied the effect of the fusion of the stem region (SR), as an N-terminal extension of the catalytic domain, on the in vitro folding efficiencies of the inclusion bodies. The stem region fused to the catalytic domain (SRCD) increases the folding efficiency of recombinant protein with native fold compared to the protein that contains only the CD. During in vitro folding, also promotes considerably the solubility of the misfolded proteins, which do not bind to UDP-agarose columns and exhibit no galactosyltransferase activity. In contrast, the misfolded proteins that consist of only the CD are insoluble and precipitate out of solution. It is concluded that a protein domain that is produced in a soluble form does not guarantee the presence of the protein molecules in a properly folded and active form. The stem domain has a positive effect on the in vitro folding efficiency of the catalytic domain of both human and bovine beta4Gal-T1, suggesting that the stem region acts as a chaperone during protein folding. Furthermore, investigation of the folding conditions of the sulphonated inclusion bodies resulted in identifying a condition in which the presence of PEG-4000 and L-arginine, compared to their absence, increased the yields of native CD and SRCD 7- and 3-fold, respectively. 相似文献