A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity

omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons. Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia. In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function. The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA. The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC). The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40 mg/L. The analgesic function was detected by using mouse hot-plate assay. After intracranially administering fusion protein with the dose of 0.6 mg/kg, marked analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6 h.     

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.     

Cloning and expression of a region of vesicle associated membrane protein2 (VAMP2) gene and its use as a recombinant peptide substrate for assaying clostridial neurotoxins in contaminated biologicals

An assay for the endopeptidase activities of clostridial neurotoxins in contaminated biotherapeutic products has been developed. Based on a synthetic peptide substrate representing amino acid residues 60–94 of the intracellular vesicle associated membrane protein2 (VAMP2), RT-PCR was used to amplify the VAMP2 sequence. The extended insert was digested with EcoRI and SalI and ligated into pGEX4T-1 vector for construction of the pGEX4T-1/VAMP plasmid for expressing in Escherichia coli a fusion protein linked to glutathione S-transferase (GST). The fusion protein was purified by affinity chromatography and used in an ELISA assay for comparison with the commercially available synthetic VAMP peptide and rabbit polyclonal antiserum. The identity of the immunoreactivity of recombinant VAMP2 protein with the chemically synthesized peptide was demonstrated by western blot. Our results indicated that recombinant VAMP2 peptide not only reacted with specific polyclonal antibody in a dose-dependent manner, without any remarkable difference observed between the reactivity of the fusion protein and commercial VAMP2 segment peptide, but also cleaved by botulinum neurotoxin type B (BONT/B) after endopeptidase assay. Thus, recombinant VAMP2 could serve as a replacement for VAMP2 synthetic peptide, potentially useful in endopeptidase assays for replacement of the currently used mouse bioassay for clostridial neurotoxins contaminating biotherapeutic products.     

Immunogenicity of the repetitive and nonrepetitive peptide regions of the divergent CS protein of Plasmodium knowlesi

The circumsporozoite (CS) protein of the Nuri strain of the simian malarial parasite Plasmodium knowlesi was expressed as a fusion protein in E. coli. This fusion protein cross-reacted with the polyclonal monkey sera raised against irradiated sporozoites of another strain (H strain) of P. knowlesi. The antibody against the repeat units of the H strain CS protein was affinity purified from the polyclonal sera by using synthetic repeat peptides. The affinity-purified antibody did not cross-react with the Nuri CS fusion protein. The immunogenicity of different regions of the CS protein was additionally studied by using several synthetic peptides. All but the most COOH-terminal peptide showed cross-reactivity with the polyclonal sera. Because the repeat regions of the CS protein of the two strains are diverse, whereas the non-repetitive regions are immunogenic and conserved, the latter may be better suited for a potential vaccine.     

重组C8orf32蛋白的表达、纯化及抗体制备

C8orf32是一种功能未知基因,其mRNA含量在乳腺癌组织中显著高于正常乳腺组织。将其开放式阅读框插入pGEX-6P1原核表达载体T7启动子控制下的GST编码基因下游,构建了C8orf32蛋白表达质粒pGEX-6P-C8。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达了GST- C8orf32融合蛋白。经带有GST标签的位点特异性蛋白酶切割除去GST-C8orf32融合蛋白的GST标签,获得了N端带有8个多余氨基酸残基的C8orf32蛋白,蛋白纯度为95%左右。N端氨基酸序列分析表明该蛋白N端氨基酸序列正确,质谱鉴定进一步证明所表达C8orf32蛋白的正确性。用制备的C8orf32蛋白免疫新西兰白兔,获得了能够正确识别C8orf32蛋白的抗血清。该蛋白及其抗体的成功制备,为进一步研究C8orf32蛋白的结构功能和体内分布打下了基础。     

大鼠转铁蛋白受体单链抗体和芋螺毒素SO3的融合蛋白在大肠杆菌中的表达

人工合成了芋螺毒素SO3的基因片段,通过链延伸方法获得了SO3的全长基因。在此基础上,将SO3基因插入到含抗大鼠转铁蛋白受体的单链抗体基因的pTIG-Trx表达载体中,构建含抗大鼠转铁蛋白受体单链抗体和SO3融合基因的重组表达载体,转化大肠杆菌BL21(DE3)和Origmia(DE3)／DsbC并得到了表达,该融合蛋白主要以包涵体形式存在。为进一步研究芋螺毒素SO3跨血脑屏障转运和治疗作用奠定了基础。     

转录因子XBP1的融合表达、纯化及多克隆抗体的制备

人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82～ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pGEX KG中 ,构建成重组质粒pGST XBP 1(82～ 14 7位氨基酸 ) .将该重组质粒转化E .coliDH5α后 ,表达GST XBP 1(82～ 14 7位氨基酸 )融合蛋白 ,经谷胱甘肽 Sepharose 4B亲和层析获得纯化的融合蛋白 .用此融合蛋白免疫家兔制备多克隆抗体 .利用制备的抗体分别用Western印迹和免疫细胞化学检测XBP 1的 2种剪切形式在哺乳动物细胞中的表达 .结果表明 ,该抗体对XBP 1的 2种剪切形式均具有反应原性 ,效价高 ,特异性好 ,可以用于进一步研究XBP 1的功能     

人GST-AWP1融合蛋白的原核表达及其抗体制备

为进一步研究人的一新蛋白———蛋白激酶C相关激酶 1相关蛋白 (AWP1)的结构、功能及与其相互作用的蛋白而进行GST AWP融合蛋白表达载体的构建、原核表达、纯化及其抗体的制备 .采用逆转录PCR(RT PCR)法从人ECV30 4内皮细胞中扩增AWP1cDNA编码区 ,并将其重组于谷胱甘肽硫转移酶 (GST)融合蛋白表达质粒pGEX KG中 .经酶切、序列鉴定分析后 ,用该重组质粒转化大肠杆菌BL2 1,并经异丙基 β D 硫代半乳糖苷 (IPTG)诱导产生GST AWP1融合蛋白 ,继而纯化获得了分子量约 5 6kD的融合蛋白 .将此融合蛋白免疫新西兰兔 ,经ELISA和Western印迹检测获得了效价高、免疫活性强的兔抗人多克隆抗体 .结果表明成功构建了GST AWP1融合蛋白表达载体 ,在大肠杆菌高效表达了GST AWP1融合蛋白 ,并获得高效多抗 ,为下阶段深入AWP1功能研究提供了重要的基础     

棉铃虫核型多角体病毒orf86基因的原核表达、抗体制备与免疫印迹检测

棉铃虫核型多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)orf86是一个功能未知的基因。从HearNPV基因组中通过PCR得到orf86基因编码序列,将其构建于原核表达载体pGEX-4T-2,转化大肠杆菌BL21获得融合表达产物。融合表达蛋白经分离纯化后免疫新西兰大白兔,制备其多克隆抗体。用ELISA测定结果表明,ORF86多克隆抗体效价为1:5.12×105。利用该抗体Western印迹检测HearNPV感染的HZAM1细胞,检测到一个36kD的目的蛋白条带,与ORF86预期大小一致。结果表明该多抗可用于对orf86编码蛋白的检测和相关功能研究。     

HIV-1 B’/C亚型Rev蛋白的表达、纯化与抗体制备

目的:为方便实验室工作中对HIV-1 B’/C亚型Rev蛋白的检测,制备相应的Rev蛋白及其抗体。方法:将我国HIV-1 B’/C亚型流行株的rev基因按大肠杆菌优势密码子进行改造后人工合成,在原核系统中与pET30a(+)载体中的His.Tag、Trx.Tag及S.Tag进行融合表达,目的蛋白经Ni2+金属螯合层析柱纯化后用于免疫家兔,制备多克隆抗体。结果与结论:合成基因在原核系统中融合表达得到相对分子质量约18×103的融合蛋白,目的蛋白的表达量约占菌体总蛋白量的36%;用纯化后的融合蛋白免疫家兔,制备了多克隆抗体,Western印迹及间接免疫荧光检测结果显示,获得的多克隆抗体与HIV-1 B’/C亚型的Rev蛋白能产生特异性反应,可用于检测HIV-1 B’/C亚型Rev蛋白的表达。     

Anti-attacin polyclonal antibody from an in vitro derived antigen used for immunoblot to quantify attacin expressed in transgenic apple

A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l^–1) by Ni²⁺-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody.     

Purification and binding features of a pea fructose-1,6-bisphosphatase domain involved in the interaction with thioredoxin f

We previously demonstrated that a cluster in the available 150 Asn-¹⁷⁰Glu region of pea chloroplast fructose-1,6-bisphosphatase (FBPase) could be involved in its interaction with the physiological modulator thioredoxin (Trx). Using as template a cDNA coding for pea chloroplast FBPase, a DNA insert coding for a 19 amino acid fragment ( 149 Pro-¹⁶⁷Gly) was amplified by PCR. After insertion in the pGEX-4T vector-1, it was expressed in Escherichia coli as a fusion protein (GST-19) with the vector-coded glutathione transferase (GST). This protein appears in the supernatant of cell lysates, and was purified to homogeneity. After thrombin digestion, the 19 amino acid insert was isolated as a polypeptide which displayed a positive reaction against pea chloroplast FBPase antibodies. GST-19 linked to glutathione-Sepharose beads, but not the GST, strongly interacts with pea Trx f , suggesting that this binding depends on the 19 amino acid insert. ELISA and Western blot experiments also demonstrate the existence of a GST-19-Trx f interaction, as well as a negligible quantity of Trx f bound by the vector-coded GST. Putative competitive inhibition assays of FBPase activity carried out in the presence of increasing concentrations of the 19 amino acid insert do not demonstrate any enzyme inhibition. On the contrary, this protein fragment enhances the enzyme activity proportionally to its concentration in the assay mixture. This indicates that the FBPase-Trx f binding promotes some type of structural modification of the Trx molecule, or of the FBPase-Trx docking site, thus facilitating the reductive modulation of FBPase.     

Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function

To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src. Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src. Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases. The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase. The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment. The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor. The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain. These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase. The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.     

一种特异性多用途抗人Connexin31抗体的获得与应用

间隙连接蛋白31(Connexin31,Cx31)是间隙连接蛋白(Connexin)家族的一员,目前对于Cx31的功能及其调节方式知之甚少。本实验利用Fmoe固相多肽合成的方法合成Cx31羧基端一个多肽片段(250-266从),经HPLC纯化后偶联到匙孔槭血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化,采用Cx31myc表达蛋白进行Western blotting、细胞免疫荧光染色、免疫沉淀实验,证实得到的抗体为抗间隙连接蛋白31的特异抗体。     

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.     

肿瘤MUC1/Y的克隆及其胞外段在大肠杆菌中的可溶性表达、纯化和鉴定

为获得MUC1 Y全长cDNA及其胞外段蛋白 ,以用于进一步的生物学功能及肿瘤生物学治疗的研究 .利用RT PCR从HeLa细胞中扩增MUC1 Y全长cDNA ;PCR扩增其胞外段 ,克隆到原核表达载体pGEX 2T ,转化DH5a菌 ,诱导表达 ;亲和层析纯化 ;凝血酶酶切、GST活性及N端蛋白测序鉴定 ;免疫家兔制备多克隆抗体 .所得MUC1 YcDNA的开放读框为 759bp ,登录于GenBank(AF12 552 5) .其信号肽编码序列缺失 9bp ,第 3 3 1位发生G A转换 ,造成缬氨酸突变为蛋氨酸 .表达获得约 4 0kD融合蛋白GST Yex ,占菌体总蛋白 2 5%～ 3 0 % ,其中 70 %～ 80 %为可溶性 ,经亲和层析一步纯化 ,纯度 >90 % ,GST比活性为 0 2 1U μg .凝血酶酶切后的N端蛋白序列测定表明与已知序列完全一致 ,抗血清ELISA效价为 1∶2 50 0 0 0 .结果表明 ,克隆到发生碱基缺失和突变的MUC1 Y全长cDNA ,获得MUC1 Y胞外段蛋白及其多抗 ,可进一步用于相关研究 .     

Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems.

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.     

Cloning and expression of feline interleukin 15

A cDNA encoding feline interleukin 15 (IL15) was cloned from the lymph node of a cat infected with feline infectious peritonitis virus. The cDNA is 486 bp in length and encodes a protein of 162 amino acids. Recombinant protein was readily expressed as a GST fusion in Escherichia coli and purified by glutathione affinity chromatography. Expression of recombinant protein in mammalian cells was only accomplished by eliminating the 5' and 3' UTR, replacing the IL15 signal peptide with the tissue plasminogen activator signal peptide, and adding 3' sequence to disrupt presumptive secondary structure of the mRNA. Biologically active feline IL15 was expressed in HEK293T cells and was shown to sustain primary feline lymphocytes, a feline T cell line, and mouse CTLL-2 cells. Proliferation of CTLL-2 cells was induced by the recombinant protein in a dose-dependent manner. Monoclonal and polyclonal antibodies against human IL15 recognized feline IL15 in immunofluorescence and Western blot assays. Additionally, feline IL15 was detectable using a commercially available human IL15 ELISA kit.     

Production of the polyclonal anti-human metallothionein 2A antibody with recombinant protein technology

Metallothionein 2A(MT2A)is a small stress response protein that can be induced by exposureto toxic metals.It is highly expressed in breast cancer cells.In this study,the cDNA encoding the humanMT2A protein was expressed as glutathione S-transferase(GST)fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separatedby electrophoresis,the recombinant protein was visualized by Coomassie blue staining and the 33 kDarecombinant GST-MT2A fusion protein band was cut out from the gel.The gel slice was minced and used togenerate polyclonal antisera.Immunization of rabbit against MT2A protein allowed the production of hightiter polyclonal antiserum.This new polyclonal antibody recognized recombinant MT2A protein in Westernblot analysis.This low-cost antibody will be useful for detection in various immuno-assays.     

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.