弗氏志贺菌5a 型 M90T 株 ClpB 蛋白在大肠杆菌中的融合表达和纯化

目的：构建弗氏志贺菌cZp8基因的原核表达质粒,在大肠杆菌中表达后,纯化带T7标签的ClpB蛋白。方法与结果：PCR扩增得到线性化表达载体pET24a与2574bp的c加曰基因片段,利用不依赖连接反应的克隆法（LIC）进行克隆,得到重组质粒pET-ClpB,转入大肠杆菌BL21（DE3）中进行诱导表达,通过一系列条件优化,确定可溶性表达条件为在30℃下、用1mmol／LIPTG诱导2h;利用抗T7单克隆抗体琼脂糖珠进行亲和纯化,得到了纯度很高的相对分子质量为95×10^3的ClpB-T7融合蛋白。结论：实现了ClpB-T7在大肠杆菌中的可溶性表达,并纯化获得了高纯度的融合蛋白。     

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.     

载脂蛋白E3的克隆及原核表达

载脂蛋白Ｅ(ａｐｏｌｉｐｏｐｒｏｔｅｉｎＥ ,ａｐｏＥ)由 2 99个氨基酸组成 ,分子量 34ｋＤ ,是维持人体正常脂质代谢的必需蛋白质 .它是乳糜微粒 (ＣＭ )、极低密度脂蛋白 (ＶＬＤＬ)和高密度脂蛋白 (ＨＤＬ)的组分 ,是极低密度脂蛋白受体的重要配体 ,是脂质进入细胞不可缺少的中介 .ａｐｏＥ有 3种同分异构体 :ａｐｏＥ2、ａｐｏＥ3、ａｐｏＥ4 ,分别具有不同的生理作用 .ａｐｏＥ4与血浆高胆固醇、心血管疾病和老年痴呆等疾病关联[1～ 3 ] .ａｐｏＥ2与Ⅲ型高脂血症有关 ,并对老年痴呆有防治作用[4,5] .ａｐｏＥ3是大多数健康人所具有…     

GST-HRB融合蛋白的表达与纯化

构建GST-HRB重组质粒,进行融合蛋白的表达、纯化及鉴定.利用PCR扩增及基因重组技术,以pcDNA-3.1-HRB为模板扩增出HRB全基因序列,并将其插入带有GST(谷胱甘肽巯基转移酶)标签的原核表达载体pGEX-6P-1中,构建GST-HRB融合蛋白表达质粒.然后,将重组质粒GST-HRB转化至大肠杆菌Rosseta进行融合蛋白的表达.利用GST琼脂糖珠进行融合蛋白的纯化,最后应用SDS-PAGE电泳和Western blotting鉴定纯化的融合蛋白.结果表明,成功构建pGEX-6P-1-HRB原核表达载体,表达及纯化了GST-HRB融合蛋白.     

High-level expression of a semisynthetic dam gene in Escherichia coli

We constructed a semisynthetic gene encoding a DNA-adenine-methyltransferase (Dam) that codes for the same amino acid sequence as the wild type (wt) Escherichia coli dam gene. Since for unknown reasons the entire wt sequence, from the start codon to the end of the gene, could not be cloned, a gene was constructed consisting of a chemically synthesized 5' portion and a 3' portion from the E. coli chromosome. Introduction of this semisynthetic gene into a suitable vector allows overproduction of E. coli Dam in mg amounts per liter E. coli culture, with optimum expression of the gene in the vector pJLA503. This plasmid places the target gene under control of the strong, tandemly arranged pR pL promoters from bacteriophage lambda, regulated by a temperature-sensitive lambda repressor. A rapid, two-column purification protocol is described that allows for very fast purification of the protein. The 32-kDa recombinant protein methylates the sequence GATC.     

TRAF6-GST融合蛋白在大肠杆菌中的表达及纯化

目的:在大肠杆菌中表达肿瘤坏死因子受体相关因子6(TRAF6)与GST的融合蛋白并进行纯化。方法:采用PCR方法从肝文库中扩增编码TRAF6的DNA片段,将其插入原核表达载体pGEX-4T-2,构建GST-TRAF6原核表达载体,并转入大肠杆菌BL21(DE3)中,用IPTG诱导表达;用谷胱甘肽-琼脂糖珠亲和纯化表达的GST-TRAF6融合蛋白。结果:酶切鉴定和测序分析显示,长为1569 bp的TRAF6 DNA片段在pGEX-4T-2-TRAF6中的碱基序列、插入位点及读框正确,且位于表达载体的GST序列下游;经IPTG最佳浓度0.5 mmol/L诱导表达、亲和纯化后,获得了相对分子质量约85×103的GST-TRAF6融合蛋白。结论:构建了重组GST-TRAF6原核表达载体,获得了GST-TRAF6的大肠杆菌BL21表达菌株及GST-TRAF6融合蛋白,利于深入研究TRAF6的功能。     

重组斑马鱼CD36蛋白的原核表达及纯化

目的：在大肠杆菌中重组表达斑马鱼CD36蛋白胞外区38～432氨基酸残基段并纯化。方法：PCR扩增斑马鱼CD36蛋白的基因编码区,连接到带有6~His标签的原核表达载体pET-28a中,构建重组表达质粒pET28a-CD36,并转化大肠杆菌BL21（DE3）,用IPTG诱导表达,优化表达条件后用Ni^2＋柱进行纯化。结果：构建了pET28a-CD36重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的46．8×10^3。结论：获得了斑马鱼CD36融合蛋白,为其生物学功能研究奠定了基础。     

Production of chimeric protein coded by the fused viral H-ras and human N-ras genes in Escherichia coli

A new method for the production of a chimeric protein of two related genes has been developed. The nucleotide sequences of the region from the N terminus to the 86th amino acid (aa) residue of human N-ras and of the Harvey sarcoma virus (Ha-MuSV) H-ras are 80% homologous. We isolated the DNA fragment encoding the N-terminal portion up to the 70th aa residue from plasmid pH-1 which encodes the total genome of Ha-MuSV, and the DNA fragment encoding the C-terminal portion from the 40th aa to the C terminus from plasmid p6a1 which includes the human N-ras cDNA but lacks the N-terminal portion. After partial digestion of both fragments with phage lambda exonuclease, which creates 3'-protruding ends, a hybrid was formed between 73% homologous single-stranded DNA portions at the 3' ends of both fragments. The hybrid was recloned on pBR322 after repairing with Escherichia coli DNA polymerase I and DNA ligase. The chimeric v-H/N-ras gene composed of the N-terminal portion of v-H-ras gene and the remaining region of N-ras gene was inserted into an expression vector containing two tandem trp promoters and a terminator, and expressed in E. coli. The chimeric protein was found to accumulate to approx. 10% of total cellular proteins.     

A comparative study of the human T-cell leukemia virus type 2 integrase expressed in and purified from Escherichia coli and Pichia pastoris

The human T-cell leukemia virus type-2 (HTLV-2) integrase (IN) catalyzes the insertion of the viral genome into the host chromosome. HTLV-2 IN was expressed as an N-terminal hexa-histidine tagged protein in the methylotrophic yeast Pichia pastoris and as a C-terminal hexa-histidine fusion in Escherichia coli. Maximal IN expression was observed at 48h post-induction for the yeast system and 2h post-induction for E. coli. Effective purification strategies were developed using non-ionic and zwitterionic detergents for initial protein extraction, followed by a one-step nickel-chelating chromatography purification. IN from both sources was routinely greater than 90% pure with yields exceeding 1.5mg of purified IN per liter of culture for P. pastoris. The relative pI was defined for both INs, pH 5.0-5.4, by 2D-gel electrophoresis. Specific activities for IN purified from E. coli and P. pastoris were calculated from in vitro 3(') processing assays and were comparable. In vitro IN assays were also performed to optimize reaction buffer pH and metal concentrations for both 3(') processing and strand transfer assays. Strand transfer was optimal from pH 6.2-6.8, more than 1.5 pH units below the optimal 3(') processing pH of 8.3. IN from both sources showed no enhancement in activity with MnCl(2) concentrations greater than 5mM. The specific activity of P. pastoris purified IN was 0.35 product (pmol)/h/microg IN, and E. coli produced IN was 0.48 product (pmol)/h/microg IN.     

重组斑马鱼 p8蛋白的原核表达及纯化

目的：在大肠杆菌中重组表达斑马鱼p8蛋白并纯化。方法：PCR扩增斑马鱼p8蛋白基因编码区,连接到带有6×His标签的原核表达载体pET-28a中,构建重组表达质粒pET-28a-p8并转化大肠杆菌BL21（DE3）,用IPTG诱导表达;优化表达条件后用Ni^2＋柱纯化重组蛋白。结果：构建了pET-28a-p8重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的12．8×10^3。结论：获得了斑马鱼p8融合蛋白,为其生物学功能研究奠定了基础。     

Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system

The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.     

日本血吸虫中国株次黄嘌呤鸟嘌呤磷酸核糖转移酶的克隆、表达及其免疫保护性研究

根据基因库中日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HGPRT) EST (BU803192) 以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjHGPRT基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接,获得SjHGPRT全长cDNA (1 270 bp),经序列分析,推断该片段含有编码SjHGPRT基因的完整阅读框,其编码基因与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SmHGPRT) 全长编码基因碱基一致性为82%,其理论推导的氨基酸组成与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶的一致性约为83%. 将其编码基因克隆到表达载体pQE30上,在大肠杆菌M15中获得准确、高效表达,表达产物分子质量约为28 ku. 用日本血吸虫成虫抗原免疫血清对表达产物进行蛋白质印迹检测,在预测位置上出现明显的识别条带. 重组蛋白动物免疫保护性结果显示：在虫荷、每克肝卵、每克粪卵和雌子宫内卵数方面,疫苗组与对照组比较差异均具有显著性 (P < 0.05,P < 0.01). 结果表明,日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SjHGPRT) 全长cDNA成功克隆并在大肠菌中得到表达,表达产物具有良好的抗原性和动物免疫保护效果,是一种潜在的具有部分免疫保护性的抗血吸虫病疫苗候选分子.     

人MCM7 N-端融合蛋白的原核表达、纯化及其与AR蛋白相互作用的研究

目的：制备人MCM7 N-端的GST融合蛋白（GST-MCM7N）,研究其是否和雄激素受体（AR）蛋白存在直接的相互作用。方法：利用RT-PCR获得长度为744 bp的人mcm7 基因N-端cDNA碱基片段,把该片段构建到原核表达载体pGEX-5x-3中,转化大肠杆菌BL-21菌株。经IPTG诱导菌株基因表达产生了GST-MCM7N融合蛋白;使用Glutathione Sepharose 4B球珠分离纯化。利用GST pull-down 技术把纯化的融合蛋白分别和前列腺癌细胞LNCaP细胞裂解液及基因工程获得的AR蛋白孵育,检测MCM7蛋白的 N-端是否和AR蛋白直接相互作用。结果：酶切鉴定及基因测序表明,mcm7 基因cDNA的 N-端片段被构建到表达载体pGEX-5x-3中;SDS-PAGE及Western blotting结果分别显示,本研究获得了GST和GST-MCM7N融合蛋白;GST pull-down 结果证实GST-MCM7N和AR蛋白存在相互作用。结论：MCM7蛋白的N-端和AR蛋白至少在体外可以发生直接的相互作用。     

Characterization of Bacillus subtilis ExoA protein: a multifunctional DNA-repair enzyme similar to the Escherichia coli exonuclease III.

To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1', 2'-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3' end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3'-5' exonuclease, ribonuclease H, and 3'-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3'-5' exonuclease activity than that of E. coli exonuclease III.     

Cloning, E. coli expression, refolding, and ATP-binding properties of a WD40-deleted Apaf-1 isoform

The apoptotic protease activating factor (Apaf-1) is central to the regulatory mechanism by which procaspase-9 is activated in the cytochrome c-mediated pathway of apoptosis. For a detailed biochemical and structural investigation of Apaf-1 function, we have cloned and expressed in Escherichia coli inclusion bodies the WD40-deleted protein (DeltaWD40 Apaf-1) from HepG2 cell. The construct contains an N-terminal His6 tag derived from the cloning vector so that the mass of the protein and the tag together is 51,594 Da, as determined by TOF/TOF mass spectrometric analysis. An optimized refolding protocol has allowed protein recovery in highly pure form. Basic fluorescence and CD probes indicate that the refolded protein retains secondary and tertiary structures, and unfolds in the presence of higher concentration of denaturant. The equilibrium ATP binding property of the protein has been measured by changes in fluorescence emission due to the fluorescent ATP analog, mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate). The results demonstrate a tight Apaf-1-ATP interaction, the binding affinity being 380 nM.     

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.     

Universal restriction site-free cloning method using chimeric primers

A universal restriction site-free cloning method has been developed to precisely insert a DNA fragment into a vector at any desired location without altering any nucleotide(s) in either the DNA fragment or the vector. The technique employs two pairs of chimeric primers, each containing a ribonucleotide. One pair of primers is used to amplify a target DNA fragment and another is used to prepare a linear vector. The ribonucleotide is used as a specific site for cleavage promoted by rare-earth metal ions such as La3+ or Lu3+. Therefore, blunt-ended PCR products can be converted into a dsDNA with single-stranded 3'overhangs for efficient ligation. The primers are designed so that both the target DNA fragment and vector PCR products create defined 3' overhangs to permit the formation of a seamless plasmid during the subsequent ligation. This method has been used successfully to clone the E. coli gene coding for peptidyl-tRNA hydrolase.     

植原体免疫主导膜蛋白Imp基因原核表达载体构建及表达

旨在构建植原体免疫主导膜蛋白Imp基因原核表达载体,并进行初步表达。以重组克隆质粒pMD18-T-Imp为模板,PCR扩增Imp基因片段。构建表达载体pET-28a(+)-Imp,转化宿主菌E.coliBL21(DE3)。筛选阳性克隆,提取重组质粒作PCR鉴定、酶切鉴定及IPTG诱导表达鉴定。PCR及双酶切结果显示,重组质粒pET-28a(+)-Imp构建成功。经IPTG诱导BL21(pET-28a(+)-Imp)表达约20 kD的蛋白,与预期的携带6×His-Tag的目的蛋白(19.5 kD)大小相符,主要以包涵体形式存在。结果显示,构建的表达载体pET-28a(+)-Imp在E.coliBL21(DE3)中能够达一定量表达,为进一步纯化Imp蛋白奠定基础。     

猪大肠杆菌耐热肠毒素和热敏肠毒素基因的融合及表达

用聚合酶链反应扩增出猪源大肠杆菌编码ST前体（proST）和LT的B亚单位(LTB)成熟多肽的序列,再通过套式PCR将proST编码序列3′端和LTB编码序列5′端融合,并置于同一阅读框内,得到ST和LTB的融合基因,将此序列克隆到pGEMT质粒中,序列分析后,亚克隆到表达载体pQE30中,在大肠杆菌细胞中得到表达,表达的融合蛋白同时具有ST和LTB的抗原性,且无ST和LT的生物毒性。     

Cloning a defined region of DNA using a limited action of DNA polymerase: application to dissection of hepatitis B virus surface antigen gene

Using dodecadeoxynucleotides as primers for DNA synthesis and 3'-o-chlorophenyl-phosphorylated dodecadeoxynucleotides as "stoppers" for chain elongation, pre-defined regions of a gene previously cloned in M13 single-stranded (ss) DNA phage were converted into double-stranded (ds) DNA utilizing the action of the Klenow fragment of Escherichia coli DNA polymerase I (PolIk). The resulting ds DNA was freed from the ss region by S1 nuclease treatment. This method can be used to obtain DNA fragments of any size with pre-defined 5' and 3' ends. About 15% of the input ss DNA template molecules are converted into ds DNA fragments. This technique was used to synthesize several DNA fragments from different portions of the hepatitis B virus surface antigen (HBsAg) gene. The products were then ligated into a yeast plasmid vector that carries the E. coli lacZ gene which is located downstream from the yeast acid-phosphatase promotor. Using this system, several fragments of HBsAg were produced in the form of beta-galactosidase fused protein.