Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures

Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.     

High-Level Expression of Rat Farnesyl:Protein Transferase inEscherichia colias a Translationally Coupled Heterodimer

Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the α- and β-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/LEscherichia coli.Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The β-subunit-coding sequence was placed upstream of the α-subunit coding sequence linked by overlapping β-subunit stop and α-subunit start codons. Additionally, the initial 88 codons of the α-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins inE. coli.Since previous attempts at recombinantly expressing FPTase inE. colifrom a translationally coupled system have demonstrated that initiation of translation of the α-subunit is poor, we propose that the optimization of the codons at the start of the α-subunit gene leads to the observed high level of recombinant expression.     

Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein

We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI? and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.     

鲑鱼降钙素基因在大肠杆菌中的克隆与表达

以鲑鱼基因组ＤＮＡ为模板,采用ＰＣＲ获得ｓＣＴ基因,并为ＤＮＡ序列分析所证实．以ｐＧＥＸ－３Ｘ为表达载体,利用体外定点突变技术成功地构建了融合蛋白ＧＳＴ－ｓＣＴ的重组表达质粒ｐＧＥＸ－３Ｘ－ｓＣＴ,在大肠杆菌中得到高效表达,其表达量约为菌体总蛋白的３０％;利用亲合层析法对融合蛋白ＧＳＴ－ｓＣＴ进行纯化,再经Ｆａｃ－ｔｏｒΧａ酶切后获得了重组ｓＣＴ,并对其进行活性检测．初步实验证明,重组ｓＣＴ具有较强的生物活性     

重组人β防御素3在大肠杆菌中的表达和活性分析

防御素是生物界广泛分布的一类低分子短肽,具有广谱高效的杀菌、抗肿瘤作用,并且不易使微生物产生抗药性,具有很高的应用价值,其中最引人注目的是β防御素[1,2].人β防御素3(humanβ-defensin3,hBD3)是最近发现的第3种人源性β防御素,与其它人防御素相比,在抗菌活性等方面具有明显优势,是所有防御素中抗菌能力最强的之一[3~7],具有独特的研究和开发价值.为了得到高效表达hBD3的工程菌株,本实验按照细菌对密码子的偏爱,人工合成了hBD3的寡核苷酸片段,构建了其表达载体.经IPTG诱导、分离纯化和肠激酶切割,得到了与天然hBD3活性基本相同的…     

Spinach holo-acyl carrier protein: overproduction and phosphopantetheinylation in Escherichia coli BL21(DE3), in vitro acylation, and enzymatic desaturation of histidine-tagged isoform I

Spinach ACP isoform I was overexpressed in Escherichia coli BL21(DE3) using a gene synthesized from codons associated with high-level expression in E. coli. The synthetic gene has extensive changes in codon usage (23 of 77 total codons) relative to that of the originally synthesized plant gene (P. D. Beremand et al., 1987, Arch. Biochem. Biophys. 256, 90-100). After expression of the new synthetic gene, purified ACP and ACP-His6 were obtained in yields of up to 70 mg L-1 of culture medium, compared to approximately 1-6 mg L-1 of purified ACP obtained from the gene composed of predicted spinach codons. In either shaken flask or fermentation culture, approximately 15% conversion to holo-ACP or holo-ACP-His6 was obtained regardless of the level of protein expression. However, coexpression of ACP-His6 with E. coli holo-ACP synthase in E. coli BL21(DE3) during pH- and dissolved O2-controlled fermentation routinely yielded greater than 95% conversion to holo-ACP-His6. Electrospray ionization mass spectrometric analysis of the purified recombinant ACPs revealed that the amino terminal Met was efficiently removed, but only if the bacterial cell lysates were prepared in the absence of EDTA. This observation is consistent with the inhibition of endogenous Met-aminopeptidase by removal of catalytically essential Co(II) and introduces the importance of considering the catalytic properties of host enzymes providing ad hoc posttranslational modification of recombinant proteins. Stearoyl-ACP-His6 was shown to be indistinguishable from stearoyl-ACP as a substrate for enzymatic acylation and desaturation. In combination, these studies provide a coordinated scheme to produce and characterize quantities of acyl-ACPs sufficient to support expanded biophysical and structural studies.     

p16抑癌基因定点突变及其在大肠杆菌中的表达与纯化

为了研究错义突变对ｐ１６功能的影响,应用ＰＣＲ体外定点突变方法对ｐ１６ｃＤＮＡ进行体外定点突变,并将野生型和突变型ｐ１６ｃＤＮＡ克隆于ｐＧＥＸ－５Ｔ载体,在大肠杆菌中经ＩＰＴＧ诱导表达,Ｗｅｓｔｅｒｎ印迹鉴定确证表达．而后用谷胱甘肽－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化野生型和突变型ｐ１６融合蛋白．得到了第４８位密码子ＣＣＧ（Ｐｒｏ）→ＣＴＧ（Ｌｅｕ）突变的ｐ１６－Ｐ４８突变体,并在大肠杆菌中表达了４２ｋＤ的ＧＳＴ－ｐ１６和ＧＳＴ－ｐ１６Ｐ４８Ｌ融合蛋白．最后经纯化得到了野生和Ｐ４８Ｌ突变的ｐ１６融合蛋白     

pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production

The production of recombinant protein in Escherichia coli is often hampered by low expression levels and low solubility. A variety of methodologies have been developed including protein production at low temperature, and fusion protein expression using soluble protein tags. Here, we present the novel cold-shock vector pCold-GST for high-level expression of soluble proteins in E. coli. This vector is a modified pCold I cold-shock vector that includes the glutathione S-transferase (GST) tag. The pCold-GST expression system developed was applied to 10 proteins that could not be expressed using conventional E. coli expression methodologies, and nine of these proteins were successfully obtained in the soluble fraction. The expression and purification of two unstable protein fragments were also demonstrated by employing a C-terminal hexa-histidine tag for purification purposes. The purified proteins were amenable to NMR analyses. These data suggest that the pCold-GST expression system can be utilized to improve the expression and purification of various proteins.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

轮状病毒VP7基因在大肠杆菌中的表达及其免疫原性

轮状病毒是世界范围内引起婴幼儿病毒性腹泻的主要病原体。VP7是轮状病毒的主要外壳蛋白和中和抗原,是发展基因工程疫苗的首选。把包含全部3个主要抗原性区域的轮状病毒SA11 VP7基因片段以谷胱甘肽S转移酶融合蛋白的形式在大肠杆菌中进行表达,表达产物占菌体总蛋白的30%左右。经一步Glutathione Sepharose4B亲和纯化,重组蛋白纯度超过90%。Western blot实验表明,重组蛋白可被抗SA11的多抗特异地识别。动物实验表明,重组抗原可在小鼠和家兔体内诱导VP7特异的抗体和一定水平的SA11中和抗体。     

Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli.

Heterologous expression of human glutathione transferase M2-2 (GST M2-2) using Escherichia coli was improved 140-fold by mutating the cDNA expressing the enzyme. Expression of GST M2-2 from this cDNA clone, pKHXhGM2, generated approximately 190 mg protein per liter of bacterial culture, corresponding to approximately 12% of the total amount of soluble protein. The high-level-expressing cDNA was generated by oligonucleotide-directed mutagenesis introducing alternative silent mutations into the third nucleotide of codons 2, 4-7, and 10-14 in the 5' end of the cDNA coding region. The choice of alternative codons was restricted to those naturally occurring in highly biased genes in E. coli. Furthermore, the wild-type TAG stop codon at the 3' end was replaced with the two stop codons TAA and TGA in tandem to increase translation termination efficiency. The resulting partially randomized cDNA library was assayed for high-level expression using immunoscreening. Sequence similarities between the constructed high-level-expressing GST M2-2 cDNA and a similarly designed cDNA encoding the closely related human GST M1-1 suggest that the codons in the region immediately following the start codon are influential in achieving high-level expression. Pyrimidines seem to be more favorable than purines in the third position of codons in optimizing the expression of these enzymes in E. coli.     

肿瘤相关抗原基因HCA520重组蛋白的表达纯化及其肝癌患者血清中相应抗体的分析

HCA5 2 0是用SEREX(serologicalidentificationofrecombinantcDNAexpressingcloning)方法 ,即用肝癌病人血清从肝癌组织cDNA表达文库中筛选得到的肝癌相关性抗原编码基因 .利用RT PCR技术检测了HCA5 2 0mRNA在各种组织中的分布情况 ,构建了GST融合表达载体并且用亲和层析的方法纯化表达的融合蛋白 .最后用Western印迹检测了重组蛋白的免疫反应性 ,用斑点印迹检测了肝癌病人血清中HCA5 2 0的天然抗体的存在情况 .结果表明 ,HCA5 2 0在各种组织中呈丰度差异分布 ,构建好的pGEX 4T 3重组载体经IPTG诱导后高效表达GST HCA5 2 0融合蛋白 ,其分子量约 4 9kD .经GST Agarose亲和层析 ,重组蛋白得到高度纯化 .Western印迹证实 ,纯化蛋白为目的重组蛋白 ,重组蛋白具有与天然蛋白相同或相似的免疫反应性 .斑点印迹分析表明 ,2 0份肝癌病人血清中有 1份HCA5 2 0抗体阳性 ,而 4份正常人均为阴性 .HCA5 2 0的足量提供 ,可用以研究其在致癌中的作用 ,并可以免疫动物制备抗体 .它作为抗原 ,分析其抗体在不同肿瘤病人中的表达情况 ,评估其在临床肿瘤诊断中的作用     

Expression and purification of the SsbB protein from Streptococcus pneumoniae

The Gram positive bacterium, Streptococcus pneumoniae, has two genes, designated ssbA and ssbB, which are predicted to encode single-stranded DNA binding proteins (SSB proteins). We have shown previously that the SsbA protein is similar in size and in biochemical properties to the well-characterized SSB protein from Escherichia coli. The SsbB protein, in contrast, is a smaller protein and has no counterpart in E. coli. This report describes the development of an expression system and purification procedure for the SsbB protein. The ssbB gene was amplified from genomic S. pneumoniae DNA and cloned into the E. coli expression vector, pET21a. Although, we had shown previously that the SsbA protein is strongly expressed from pET21a in the E. coli strain BL21(DE3)pLysS, no expression of the SsbB protein was detected in these cells. However, the SsbB protein was strongly expressed from pET21a in the Rosetta(DE3)pLysS strain, a derivative of BL21(DE3)pLysS which supplies the tRNAs for six codons that are used infrequently in E. coli. The differential expression of the two SSB proteins in the parent BL21(DE3)pLysS strain was apparently due to the presence of two rare codons in the ssbB gene sequence that are not present in the ssbA sequence. Using the Rosetta(DE3)pLysS/pETssbB expression system, a protocol was developed in which the SsbB protein was purified to apparent homogeneity. DNA binding assays confirmed that the purified SsbB protein had single-stranded DNA binding activity. The expression and purification procedures reported here will facilitate further investigations into the biological role of the SsbB protein.     

Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins.

Previous studies have shown that the 30S ribosomal subunit of Escherichia coli can be reconstituted in vitro from individually purified ribosomal proteins and 16S ribosomal RNA, which were isolated from natural 30S subunits. We have developed a 30S subunit reconstitution system that uses only recombinant ribosomal protein components. The genes encoding E. coli ribosomal proteins S2-S21 were cloned, and all twenty of the individual proteins were overexpressed and purified. Reconstitution, following standard procedures, using the complete set of recombinant proteins and purified 16S ribosomal RNA is highly inefficient. Efficient reconstitution of 30S subunits using these components requires sequential addition of proteins, following either the 30S subunit assembly map (Mizushima & Nomura, 1970, Nature 226:1214-1218; Held et al., 1974, J Biol Chem 249:3103-3111) or following the order of protein assembly predicted from in vitro assembly kinetics (Powers et al., 1993, J MoI Biol 232:362-374). In the first procedure, the proteins were divided into three groups, Group I (S4, S7, S8, S15, S17, and S20), Group II (S5, S6, S9, Sll, S12, S13, S16, S18, and S19), and Group III (S2, S3, S10, S14, and S21), which were sequentially added to 16S rRNA with a 20 min incubation at 42 degrees C following the addition of each group. In the second procedure, the proteins were divided into Group I (S4, S6, S11, S15, S16, S17, S18, and S20), Group II (S7, S8, S9, S13, and S19), Group II' (S5 and S12) and Group III (S2, S3, S10, S14, and S21). Similarly efficient reconstitution is observed whether the proteins are grouped according to the assembly map or according to the results of in vitro 30S subunit assembly kinetics. Although reconstitution of 30S subunits using the recombinant proteins is slightly less efficient than reconstitution using a mixture of total proteins isolated from 30S subunits, it is much more efficient than reconstitution using proteins that were individually isolated from ribosomes. Particles reconstituted from the recombinant proteins sediment at 30S in sucrose gradients, bind tRNA in a template-dependent manner, and associate with 50S subunits to form 70S ribosomes that are active in poly(U)-directed polyphenylalanine synthesis. Both the protein composition and the dimethyl sulfate modification pattern of 16S ribosomal RNA are similar for 30S subunits reconstituted with either recombinant proteins or proteins isolated as a mixture from ribosomal subunits as well as for natural 30S subunits.     

Purification and characterization of carbonic anhydrase of rice (Oryza sativa L.) expressed in Escherichia coli

Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).     

链霉菌来源的UDP-葡萄糖-4-差向异构酶基因的克隆表达及酶性质研究

经同源性比较,链霉菌139(Streptomycessp.139)产生胞外多糖依博素的生物合成基因簇中ste19基因编码的蛋白Ste19与UDP_葡萄糖_4_差向异构酶有较高同源性。将ste19基因克隆至质粒pET30a,在大肠杆菌BL21(DE3)中进行了异源表达。产生的可溶性Ste19重组蛋白,占细胞总蛋白的26%,说明该基因高GC含量(73.8%)及第三位碱基偏向使用GC(96.2%)并未影响其高效表达。SDS_PAGE结果显示重组蛋白的分子量约37kD,与理论推测值基本相同。经亲和层析纯化后得到了较高纯度的重组蛋白,经HPLC分析纯度为92.9%。酶活性分析表明:Ste19蛋白可将UDP_葡萄糖转化为UDP_半乳糖,因此,Ste19蛋白是UDP_葡萄糖_4_差向异构酶,它可能参与了依博素的生物合成。     

High-level expression of the recombinant hybrid peptide cecropinA(1-8)-magainin2(1-12) with an ubiquitin fusion partner in Escherichia coli

The hybrid antibacterial peptide CA-MA [cecropinA(1-8)-magainin2(1-12)] with potent antimicrobial properties but no hemolytic activity is a potential alternative antibiotic. To explore a new approach for high-level expression of the hybrid peptide CA-MA in Escherichia coli, the sequence of ubiquitin (UBI) from housefly was inserted into the plasmid pQE30 to construct the vector pQEUBI. The cDNA fragment encoding CA-MA with preferred codons of E. coli was obtained by recursive PCR (rPCR) and cloned into the vector pQEUBI to express the fusion protein (His)(6)-UBI-CA-MA. The fusion protein was expressed in soluble form under the optimized conditions at high level (more than 36% of the total proteins). With (His)(6)-tag, the fusion protein was easily purified by Ni-NTA chromatography and 36 mg of fusion protein was purified from 1L of culture medium. The fusion protein was efficiently cleaved by ubiquitin C-terminal hydrolase (UCH), yielding recombinant CA-MA with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant CA-MA was purified to homogeneity by reversed-phase HPLC and 6.8mg of pure active CA-MA was obtained from 1L culture medium. Analysis of recombinant CA-MA by MALDI-TOF-MS showed that the molecular weight of the purified recombinant CA-MA was 2559Da, which perfectly matches the mass (2559Da) calculated from the amino acid sequence. Analysis of CA-MA by circular dichroism (CD) revealed that the secondary structures of CA-MA in water solution were 17.4% alpha-helix and 82.6% random coil but no beta-sheet. Our results demonstrated that functional CA-MA can be produced in sufficient quantities using the ubiquitin fusion technique. This is the first report on the heterologous expression of a hybrid antibacterial peptide fused to ubiquitin in E. coli.     

Ribosome can resume the translation in both +1 or -1 frames after encountering an AGA cluster in Escherichia coli

In Escherichia coli the rare codons AGG, AGA and CGA are reported to have a detrimental effect on protein synthesis, especially during the expression of heterologous proteins. In the present work, we have studied the impact of successive clusters of these rare codons on the accuracy of mRNA translation in E. coli. For this purpose, we have analyzed the expression of an mRNA which contains in its 3' region a triplet and a tandem of AGA codons. This mRNA is derived from the human hepatitis B virus (HBV) preC gene. Both in eukaryotic cells and in eukaryotic cell-free translation system, this mRNA, directs the synthesis of a single 25 kDa protein. However, in a conventional E. coli strain BL 21 (DE3), transformed with a plasmid expressing this protein the synthesis of four polypeptides ranging from 30 to 21.5 kDa can be observed. Using different approaches, notably expression of i) precore mutated proteins or ii) chimeric proteins containing HA- and Myc-tags downstream of the AGA clusters (respectively in the -1 or +1 frame), we have found that when the ribosome encounters the AGA clusters, it can then resume the translation in both +1 and -1 frames. This result is in agreement with the model proposed recently by Baranov et al. (Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10, 221-230), thus confirming that AGA/AGG codons can serve as sites for -1 frameshifting events. Only +1 frameshifting was suggested previously to occur at the AGA/AGG clusters.     

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.