Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase.

Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.     

Zinc-binding property of the major yolk protein in the sea urchin - implications of its role as a zinc transporter for gametogenesis

Major yolk protein (MYP), a transferrin superfamily protein that forms yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP in the coelomic fluid (CFMYP; 180 kDa) has a higher molecular mass than MYP in eggs (EGMYP; 170 kDa). Here we show that MYP has a zinc-binding capacity that is diminished concomitantly with its incorporation from the coelomic fluid into the gonad in the sea urchin Pseudocentrotus depressus. Most of the zinc in the coelomic fluid was bound to CFMYP, whereas zinc in eggs was scarcely bound to EGMYP. Both CFMYP and EGMYP were present in nutritive phagocytes, where CFMYP bound more zinc than EGMYP. Saturation binding assays revealed that CFMYP has more zinc-binding sites than EGMYP. Labeled CFMYP injected into the coelom was incorporated into ovarian and testicular nutritive phagocytes and vitellogenic oocytes, and the molecular mass of part of the incorporated CFMYP shifted to 170 kDa. Considering the fact that the digestive tract is a major production site of MYP, we propose that CFMYP transports zinc, essential for gametogenesis, from the digestive tract to the ovary and testis through the coelomic fluid, after which part of the CFMYP is processed to EGMYP with loss of zinc-binding site(s).     

A novel protocol for the production of recombinant LL-37 expressed as a thioredoxin fusion protein

LL-37 is the only cathelicidin-derived antimicrobial peptide found in humans and it has a multifunctional role in host defense. The peptide has been shown to possess immunomodulatory functions in addition to antimicrobial activity. To provide sufficient material for biological and structural characterization of this important peptide, various systems were developed to produce recombinant LL-37 in Escherichia coli. In one previous approach, LL-37 coding sequence was cloned into vector pET-32a, allowing the peptide to be expressed as a thioredoxin fusion. The fusion protein contains two thrombin cleavage sites: a vector-encoded one that is 30-residue upstream of the insert and an engineered one that is immediately adjacent to LL-37. Cleavage at these two sites shall generate three fragments, one of which is the target peptide. However, when the fusion protein was treated with thrombin, cleavage only occurred at the remote upstream site. A plausible explanation is that the thrombin site adjacent to LL-37 is less accessible due to the peptide's aggregation tendency and cleavage at the remote site generates a fragment, which forms a large aggregate that buries the intended site. In this study, I deleted the vector-encoded thrombin site and S tag in pET-32a, and then inserted the coding sequence for LL-37 plus a thrombin site into the modified vector. Although removing the S tag did not change the oligomeric state of the fusion protein, deletion of the vector-encoded thrombin site allowed the fusion to be cleaved at the engineered site to release LL-37. The released peptide was separated from the carrier and cleavage enzyme by size-exclusion chromatography. This new approach enables a quick production of high quality active LL-37 with a decent amount.     

A Zinc Site in the C-Terminal Domain of RAG1 Is Essential for DNA Cleavage Activity

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn²⁺ ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn²⁺ ions bound to the catalytically active core region of RAG1 under various conditions. Using metal quantitation methods, we determined that core RAG1 can bind up to four Zn²⁺ ions. Stripping the full complement of bound Zn²⁺ ions to produce apoprotein abrogated DNA cleavage activity. Moreover, even partial removal of zinc-binding equivalents resulted in a significant diminishment of DNA cleavage activity, as compared to holo-Zn²⁺ core RAG1. Mutants of the intact core RAG1 and the isolated core RAG1 domains were studied to identify the location of zinc-binding sites. Significantly, the C-terminal domain in core RAG1 binds at least two Zn²⁺ ions, with one zinc-binding site containing C902 and C907 as ligands (termed the CC zinc site) and H937 and H942 coordinating a Zn²⁺ ion in a separate site (HH zinc site). The latter zinc-binding site is essential for DNA cleavage activity, given that the H937A and H942A mutants were defective in both in vitro DNA cleavage assays and cellular recombination assays. Furthermore, as mutation of the active-site residue E962 reduces Zn²⁺ coordination, we propose that the HH zinc site is located in close proximity to the DDE active site. Overall, these results demonstrate that Zn²⁺ serves an important auxiliary role for RAG1 DNA cleavage activity. Furthermore, we propose that one of the zinc-binding sites is linked to the active site of core RAG1 directly or indirectly by E962.     

Families of metalloendopeptidases and their relationships.

Crystal structures available for four metalloendopeptidases have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in metalloendopeptidases. A scheme based on the zinc-binding site is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidines and one glutamate are zinc-ligands in the thermolysin family. Three histidines and one tyrosine are zinc ligands in the other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyrosine.     

化学合成虎纹捕鸟蛛毒素-I基因的克隆和表达

本文报道了全化学合成虎纹捕鸟蛛毒素－Ⅰ基因在大肠杆菌中的表达,表达产物为Ｎ－端是谷胱甘肽硫转移酶的融合蛋白．经ＧＳＨ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化,凝血酶酶解融合蛋白,得到重组ＨＷＴＸ－Ⅰ（ｒＨＷＴＸ－Ⅰ）．质谱和氨基酸顺序分析均表明ｒＨＷＴＸ－Ⅰ系正确表达产物．还原复性的ｒＨＷＴＸ－Ⅰ表现出与天然ＨＷＴＸ－Ⅰ生物学活性的一致性．     

Efficient expression of truncated recombinant cadmium-metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis in Escherichia coli

Truncated recombinant metallothionein GST–fusion protein has been successfully expressed in Escherichia coli. The previously identified novel Cd-inducible metallothionein (TMCd1) gene from the locally isolated ciliate, Tetrahymena tropicalis lahorensis, was inserted into a pET-41a vector, in frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail. Truncated recombinant GST fusion protein has been purified by affinity column chromatography using glutathione sepharose. After enzymatic cleavage of GST tail with enterokinase, the truncated TMCd1 MT shows molecular weight of 11.5 kDa, corresponding to the expected value. This is the first successful report of expression of cadmium metallothionein gene of a ciliate, T. t. lahorensis, reported from this part of the world, in E. coli. This study will further help in characterization of metallothionein protein of this ciliate.     

Fusion expression of bovine lactoferricin in Escherichia coli

The drug resistance problem has been growing with the utilization of current antibiotics in feed and medical industries. LfcinB, a 25-amino acid antibacterial peptide derived from bovine lactoferrin, is one of potential alternatives of antibiotics. According to the bias of codon utilization of Escherichia coli, a fragment encoding LfcinB has been chemically synthesized, inserted into vector pGEX-4T-2 and expressed in E. coli. The antibacterial peptide was fused with GST with a protease cleavage site located between them. Two constructs with different cleavage sites were made. One construct, pGEX-Th-LfcinB, contains a thrombin cleavage site carried by the vector, and the other, pGEX-Th-Xa-LfcinB, contains a Factor Xa cleavage site which was introduced after the thrombin cleavage site. Fusion protein GST-Th-LfcinB protein was efficiently cleaved by thrombin, yielding recombinant LfcinB showing antibacterial activity. However, fusion protein GEX-Th-Xa-Lfcin B containing Factor Xa recognition site could not be cleaved by Factor Xa at the conditions tried in this study. Successful expression of LfcinB in E. coli provides a possible method to produce LfcinB in large amounts.     

组织因子膜外区融合蛋白基因的表达及产物的分离纯化与活性分析

 为构建表达组织因子 (TF)膜外区的融合载体 ,制备组织因子膜外区 ,抽提人胎盘组织的总RNA,通过 RT- PCR法扩增出 TF的 c DNA克隆至 p UC1 8并测定全序列 .然后以此为模板 ,再次PCR扩增出 TF膜外区 (soluble TF,s TF) c DNA,并将其插入到谷胱甘肽巯基转移酶融合表达载体 p GEX4T- 1 ,构建了 tac启动子控制下的 GST- s TF融合蛋白的表达载体 .表达的融合蛋白经亲和层析、凝血酶切得到纯化的 s TF.表达产物经 ELISA验证 ,能特异性地与 TF抗体结合 .重新脂化后 ,该产物具有较大凝血活性 .以上说明采用融合蛋白表达系统可以大量制备组织因子膜外区 ,为研制国产重组凝血活酶试剂和研究 s TF的结构和功能创造条件 .     

Recombinant GST-human osteopontin fusion protein is functional in RGD-dependent cell adhesion

Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells. Native OPN has been shown to support cell adhesion in an RGD-peptide-inhibitable fashion. Here we expressed human OPN in E. coli as a recombinant fusion protein with glutathione-S-transferase (GST). We report that the GST-OPN fusion protein has functional activity. PAP2 (ras-transformed, metastatic murine NIH 3T3) and MDA-MB-435 human mammary carcinoma cells bound to GST-OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN. Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the recombinant and native OPN proteins by similar, integrin-mediated mechanisms. Adhesion to both sources of OPN also was inhibited by thrombin treatment of the protein. Thrombin cleaves GST from OPN in the fusion protein, and also cleaves internally in OPN, adjacent to the RGD sequence of the protein. Our results suggest that (a) thrombin cleavage of native OPN may be a natural regulator of OPN function, and (b) the majority of OPN cell binding activity is mediated by the RGD sequence in the protein backbone, with little or no requirement for post-translational modifications that occur in native OPN for adhesive function as measured here.     

pC6-2/caspase-6 system to purify glutathione-S-transferase-free recombinant fusion proteins expressed in Escherichia coli

Glutathione-S-transferase (GST) fusion protein expression vectors are often employed for the expression and purification of proteins in Escherichia coli. GST is then removed by site-specific proteolysis using thrombin. However, the presence of internal thrombin cleavage sites in expressed proteins can severely affect the purification of intact proteins. Cysteine-dependent aspartate-specific proteases (caspases) are efficient enzymes with defined substrate specificity. Unlike most of the proteases used for the removal of affinity tags, caspases do not leave any amino acids at the amino-terminus of cleaved proteins. We have engineered the caspase-6 site VEMD in a pGEX vector to give the pC6-2 vector. The caspase-6 can be easily removed after cleavage. Here, we describe the detailed protocol for purifying proteins using our pC6-2/caspase-6 expression and purification system. The cleavage by caspase-6 occurs in <30 min and the entire procedure can be completed in 2 d.     

Expression, purification, and characterization of recombinant human beta-amyloid42 peptide in Escherichia coli

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Evidence indicates that abnormal processing and extracellular deposition of the beta-amyloid42 peptide, the longer form of proteolytic derivative of the transmembrane glycoprotein-amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Since it is convenient and economical to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify beta-amyloid42 using glutathione-S-transferase (GST) fusion system. beta-Amyloid42 gene was inserted into a vector pGEX-4T-1 to construct a GST-fusion protein. The fusion protein GST-beta-amyloid42, expressed in BL21 (DE3) strain, was purified with GSH-affinity chromatography followed by thrombin cleavage. The digested product was further purified with an additional GSH-affinity and a Benzamidine chromatography step. After cleavage and purification, the beta-amyloid42 moiety showed the expected size of 4.5 kDa on Tricine-SDS-PAGE, and was further confirmed by Western blot. Moreover, the fibrillar recombinant beta-amyloid42 exhibited great aggregation activity and showed neurotoxicity on neuron cells in vitro. These results suggest that our method will be useful in obtaining a large quantity of recombinant beta-amyloid42 peptide for further physiological and biochemical studies.     

高效原核表达载体pBV220的改造与应用

以高效原核表达载体 pBV220为骨架载体,应用单链寡核苷酸引物插入法在pBV220多克隆位点的下游插入了六聚组氨酸融合标签编码序列(6×His-Tag)、羟胺和凝血酶蛋白切割位点, 并增加了Xho I和Kpn I酶切位点和强终止密码子TAA, 将此新质粒命名为 pBV223。以此载体表达的目的蛋白在羧基端(C端)带有六聚组氨酸尾以利于通过固定化金属亲和层析快速纯化目的蛋白, 酶切及核苷酸序列分析验证了我们的设计。将终止密码缺失突变的nm23-H1 cDNA克隆入pBV223载体中, 在大肠杆菌DH5a中成功地表达了Nm23-H1蛋白, 通过镍(Ni)亲和层析一步即简单、快速地得到了纯化蛋白。我们所应用的单链寡核苷酸引物插入直接进行定向克隆的方法是目前为止最简便的方法。     

The astacin family of metalloendopeptidases

Molecular cloning of a human intestinal brush border metalloendopeptidase (N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase, PPH) and a mouse kidney brush border metalloendopeptidase (meprin A) has revealed 82% identity in the NH2-terminal amino acid sequences (198 residues) of the mature enzymes. Furthermore, searching of protein sequence data bases with the inferred peptide sequences as probes revealed strong similarities to astacin, a crayfish digestive protease, and an NH2-terminal domain of a human bone morphogenetic protein (BMP-1). Meprin A and PPH both have approximately 30% identity with astacin and BMP-1. Multiple alignment analysis indicated that 37 residues, including 3 cysteine residues, are strictly conserved for the four proteins in a sequence frame equivalent to the complete 200-amino acid astacin sequence. The four proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases, within an extended sequence of HEXXHXXGFXHE which is unique to this subgroup of metalloendopeptidases. In addition, the four proteins have 54% identity in a 24-amino acid sequence that includes the putative active site. A fifth protein, Xenopus laevis developmentally regulated protein UVS.2, also shares sequence identity with the metalloendopeptidases. These data provide strong evidence for an evolutionary relationship of these proteins. It is suggested that this new family of metalloendopeptidases be called the "astacin family."     

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 +/- 0.0013, 0.0095 +/- 0.0016, and 0.0080 +/- 0.0006 mg, respectively, to 50 microL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 +/- 0.0043, 0.0219 +/- 0.0035, and 0.0190 +/- 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.     

重组FAS分子的表达、纯化及抗体制备

用ＰＣＲ法扩增出编码人ＦＡＳ分子胞外区的ｃＤＮＡ片段,直接克隆到ｐＧＥＭ－Ｔ载体上,经ＤＮＡ序列测定后,再插入到谷胱甘肽转硫酶（ＧＳＴ）融合蛋白表达载体ｐＧＥＸ－ＫＧ的ＥｃｏＲⅠ和ＳａｌⅠ位点之间,构成重组质粒ｐＫＧ－ｈＦＡＳ,将此质粒导入大肠杆菌,经ＩＰＴＧ诱导后获得ＧＳＴ－ｈＦＡＳ重组融合蛋白的表达,用谷胱甘肽偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ经亲合层析获得纯化的ＧＳＴ－ｈＦＡＳ蛋白,经凝血酶酶切和二次亲合层析去除ＧＳＴ部分,得到纯化的ＦＡＳ蛋白．用纯化的ＦＡＳ抗原免疫家兔制备了抗ＦＡＳ抗体,经检测发现抗ＦＡＳ抗体能诱导Ｕ９３７细胞发生细胞凋亡     

幽门杆菌Catalase/GST融合蛋白的表达、标签切除及鉴定

旨在利用GST融合基因表达系统表达幽门螺杆菌Catalase融合蛋白,并利用凝血酶切除GST标签.将重组质粒Catalase/pGEX-4T-1转化大肠杆菌BL21( DE3)感受态中,用IPTG进行诱导表达,菌体经反复冻融、溶菌酶裂解及超声破菌后,Catalase/GST融合蛋白以部分可溶性的形式表达在上清中.采用谷胱甘肽琼脂糖树脂Glutathione Sepharose 4B对其进行纯化,得到Catalase/GST融合蛋白,再用凝血酶进行GST标签的切除,所得产物进行Western blotting鉴定.高效表达出Catalase/GST融合蛋白的相对分子质量约85 kD,凝血酶成功地切除了GST标签,Western blotting证实Catalase蛋白能被鼠抗Catalase单克隆抗体识别.     

New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.