摘要
将简单节杆菌3-甾酮-1-脱氢酶基因(ksdD)克隆到大肠杆菌表达载体pET-22b(+)上,构建重组质粒pET-ksdD,利用IPTG诱导酶在大肠杆菌BL21(DE3)中的表达,4h后取样,分别对超声破碎后的上清和沉淀进行SDS-PAGE和酶活分析,结果表明表达出的3-甾酮-1-脱氢酶的分子量大约为56kD,超声破碎后的细胞破碎液对4-AD的转化率较简单节杆菌提高了近10倍。
3-Ketosteroid △^1-Dehydrogenase gene （ksdD）of Arthrobacter Simplex was cloned into expression vector pET-22b （＋）constituting recombination vector pET-ksdD which was transfurmed into BL21 （DE3）. Positive transformant was cultivated and IPTG was added into culture to induce expression of 3-Ketosteroid-△^1-Dehydrogenase. Affter 4h induction,the supematant and sedimentation after sonic disruption were analyzed by SDS-PAGE,and were used for in vitro steroid transformation test. The result indicated 3-Ketosteroid-△^1-Dehydrogenase was expressed in E.coli successfully. The molecular weight of expressed protein was about 56kD. The transformation rate of 4-AD by cell extracts of E.coli recombinants improved 10 folders than the extracts of Arthrobacter simplex.
出处
《生物技术通报》
CAS
CSCD
2008年第3期115-118,共4页
Biotechnology Bulletin
关键词
3-甾酮-1-脱氢酶
大肠杆菌
表达
甾体转化
3-Ketosteroid △^1-Dehydrogenase E.eoli Expression Steroid transformation
