带锚链的甲基对硫磷水解酶的基因构建及表达纯化

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0.4mmol· L-1的IPTG在30℃下诱导表达5h,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 re-combinant plasmid pET28a-mph-lc was constructed and expressed in E. Coli BL2KDE3). 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-' lasted for 5 h at 30℃ when OD6OO was 0. 5~0. 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.