赤红球菌JDM312环己酮单加氧酶的原核表达及检测

Prokaryotic Expression and Detection of Cyclohexanone Monooxygenase from Rhodococcus ruber JDM312

通过酶切法从重组质粒pUC18-chnB中得到编码环己酮单加氧酶的chnB基因序列,将其定向插入原核表达载体pQE30中,构建重组质粒pQE30-chnB,转化到大肠杆菌BL21中并诱导表达目的蛋白,用SDS-PAGE电泳检测表达产物.测序结果表明chnB基因大小为1 623bp,编码由540个氨基酸残基构成的多肽.SDS-PAGE结果显示,表达分子量约为60kDa的带有6×His标签的环己酮单加氧酶,与预期分子量相符,表明成功构建出原核表达质粒并实现了目的蛋白表达,为进一步开展环己酮单加氧酶活性研究奠定了基础.

The chnB gene encoding a cyclohexanone monooxygenase was isolated from the recombinant plasmid pMD18-chnB by digested with BamHI and HindⅢ,then cloned into the expression vector pQE30 and expressed in E.coli BL21 by IPTG induction.The expressed protein was identified by SDS-PAGE.The sequence analyses showed that the coding region of the chnB of Rhodococcus ruber JDM312 was 1 623 bp in length,and the predicted primary protein was composed of 540 amino acids.The SDS-PAGE analyses revealed a single protein band with a molecular weight of 60 kDa,suggesting that the recombinant plasmid of pQE30-chnB was successfully constructed and the target protein was expressed in E.coli.