gfp融合的甲基对硫磷水解酶基因mph在E. coli中的表达

The expression of methyl parathion hydrolase fusion with gfp in E. coli

依照大肠杆菌密码子的偏爱性将来源于Pseudomonas sp. M6的有机磷水解酶基因mph设计合成了新的有机磷水解酶基因,然后将其与GFP融合进行表达,构建一株具有全细胞催化活性的大肠杆菌工程菌.设计优化后所合成的mph-r基因全长927 bp,然后将基因克隆到p ET23a-gfp大肠杆菌表达型质粒上,成功构建了重组表达质粒p ET23a-gfpmph-r,转化大肠杆菌感受态Rosetta blue.经过诱导剂IPTG的低温诱导24 h后,收集细胞,通过SDS-PAGE和Western Blot检测融合蛋白的表达.实验结果显示,融合蛋白的表达量大大提高,且通过酶活分析测定,全细胞酶活达到了11. 2 U/m L.

According to the codon preference of Escherichia coli,a new organophosphorus hydrolase gene(mph)from Pseudomonas sp.M6 was designed and synthesized,and then was fused at the C-terminal of GFP to construct E.coli engineering bacteria with whole-cell catalytic activity.The optimized mph-r gene was 927 bp in length and cloned into pET23a-gfp expression vector.The recombinant plasmid pET23a-gfp-mph-r was successfully constructed and transformed into Escherichia coli Rosetta blue.The results showed that the expression of fusion protein was greatly enhanced by SDS-PAGE and western blot assay after induced for 24 hours by adding IPTG,and the activity of whole cell reached 11.2 U/mL by enzymatic activity assay.