脱氢奎尼酸合成酶基因aroB在大肠杆菌中的克隆表达及酶活力测定

Cloning and Expression of Dehydroquinate Synthase aroB Gene in Escherichia coli

目的:在大肠杆菌中高效表达脱氢奎尼酸合成酶。方法:以大肠杆菌基因组为模板,采用PCR扩增得到脱氢奎尼酸合成酶基因aroB,并在大肠杆菌中进行表达。结果:在浓度为0.5 mmol/L的IPTG诱导4 h后aroB基因表达量占菌体总可溶性蛋白40.0%,SDS-PAGE显示重组菌诱导后表达的脱氢奎尼酸合成酶蛋白分子量约为38 kDa,发酵液中脱氢奎尼酸合成酶的酶活力达到178 U/L,为宿主菌的35.6倍。结论:脱氢奎尼酸合成酶在大肠杆菌中实现了高效表达。

AIM:To highly express dehydroquinate synthase in E.coli.METHODS:The aroB gene of dehydroquinate synthase from the genome of E.coli was amplified by PCR and expressed in E.coli.RESULTS:After being induced with 0.5 mmol/L IPTG for 4 h,dehydroquinate synthase accounted for 40.0% of the total soluble proteins with the molecular weight of 38 kDa determined by SDS-PAGE.The dehydroquinate synthase activity of the genetic engineering strain E.coli BL21/pETDuet-aroB was 178 U/L,which was 35.6 folds of the host.CONCLUSION:Dehydroquinate synthase was highly expressed in E.coli.