青蒿法呢醇合酶原核表达、纯化与功能鉴定

Escherichia coli Expression,Purification and Functional Identification of Farnesol Synthase from Artemisia annua L.

将经RACE方法克隆到的青蒿倍半萜合酶cDNA(AF304444)开放阅读框插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间,构建N端和C端均携带有HIS6表达标签的重组表达载体pET30SESQ。将pET30SESQ转入大肠杆菌BL21(DE3),IPTG(Isopropyl-beta-D-thiogalactoside)诱导蛋白表达,表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系(FPP),GC-MS分析酶促反应体系的正己烷萃取物,结果显示此重组酶可以催化FPP向法呢醇的转化。

The open reading frame of sesquiterpene cyclase cDNA（AF304444） from Artemisia annua was subcloned into a bacterial expression vector pET30a（＋） in frame with N-terminal and C-terminal HIS6-tag,then recombinant vector pET30SESQ was introduced into Escherichia coli strain BL21（DE3）.Recombinant sesquiterpene cyclase was induced at 28℃ by adding 0.5 mmol/L IPTG（Isopropyl-beta-D-thiogalactoside） and purified using immobilized metal affinity chromatography on Ni2＋ columns.GC-MS analysis showed that the recombinant sesquiterpene cyclase can catalyze the formation of farnesol from FPP.