带锚链的甲基对硫磷水解酶的基因构建及表达纯化  

Recombinant Expression and Purification of A Novel Methyl Parathion Hydrolase

作　　者：刘立坚[1] 冷艳[2] 李雨亭[1] 谢卫红[1] 

机构地区：[1]湖北工业大学生物工程学院发酵工程省部共建教育部重点实验室,湖北武汉430068 [2]中国科学院武汉病毒研究所病毒学国家重点实验室,湖北武汉430071

出　　处：《化学与生物工程》2011年第7期26-28,66,共4页Chemistry & Bioengineering

基　　金：国家自然科学基金资助项目(20875024/B0509);湖北省教育厅重大研究项目(Z20081402)

摘　　要：用PCR方法获得甲基对硫磷水解酶（MPH）编码基因,构建了重组表达质粒pET28a-mph-lc,将其转化至大肠杆菌BL21（DE3）中进行表达。当OD600达到0.5~0.6时,用终浓度0.4 mmol·L-1的IPTG在30℃下诱导表达5 h,破碎离心后,利用Ni-NTA亲和层析纯化得到具有活性的融合蛋白MPH-L,即在甲基对硫磷水解酶的C端连上了一段末端为半胱氨酸的锚链。The encoding region of mph gene for methyl parathion hydrolase was subcloned by PCR.The recombinant plasmid pET28a-mph-lc was constructed and expressed in E.coli BL21（DE3）.It was found that the fusion protein was expressed in bulk when the culture induced by isopropyl-β-D-galatose with final concentration of 0.4 mmol·L-1 lasted for 5 h at 30℃ when OD600 was 0.50.6.The pure recombinant protein MPH-L which remained its catalytic activity was obtained by purification using His-Tag nickel affinity chromatography.Therefore,methyl parathion hydrolase（MPH） was modified with a linker peptide and cysteine（Cys） in sequence at its C-terminal to form a fusion structure by gene manipulation.

关 键 词：甲基对硫磷水解酶 基因修饰 蛋白表达纯化 

分 类 号：Q786[生物学—分子生物学]