文献类型：期刊文献

中文题名：大肠杆菌caiE基因的克隆与高效表达

英文题名：Cloning and Expression of Escherichia coli cai E Gene

作者：范立强[1];袁勤生[1];吴祥甫[2]

机构：[1]华东理工大学生物反应器工程国家重点实验室生物化学研究所,上海200237;[2]中国科学院上海生物化学研究所,上海200031

年份：2002

卷号：28

期号：2

起止页码：149

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;EI(收录号：2002397116934);Scopus;北大核心:【北大核心2000】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：肉碱;表达载体;基因克隆;基因表达;大肠杆菌

外文关键词：carnitine; expression vector; gene cloning; gene expression; E.coli

摘要：用聚合酶链反应 ( PCR)从大肠杆菌 K1 2 s中扩增大肠杆菌肉碱代谢相关酶基因 cai E,将其克隆到克隆载体 p Bluescript SK中 ,测序表明 :该基因有 61 2 bp,与文献报道相比 ,有 1 0个核苷酸不同 ,相应的翻译氨基酸有 6个相异。将该基因重组到 Col E1为复制子、T7为启动子控制下的分泌型表达载体 p ET- 2 2 b( + )中 ,构建表达质粒 p ETCai E;重组到载体 p ACYC1 84中构建以 p1 5 A为复制子、Lac为启动子的表达质粒 p ACYC- Cai E;重组到载体 p WSK1 2 9中 ,构建以 p SC1 0 1为复制子、T7为启动子的表达质粒 p SC- Cai E。上述表达质粒转化大肠杆菌 BL2 1 ( DE3) ,经 1 mmol/L异丙基硫代 -β- D-半乳糖苷 ( IPTG)诱导后 ,均可表达。 SDS- PAGE分析表明表达蛋白分子质量约2 6ku,表达量占菌体总蛋白质的比例分别为 p ETCai E30 % ,p ACYC- Cai E8% ,p SC- Cai E5 %
The cai E gene, which encodes an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine recemase activities, was first iso lated by PCR from a genomic library of E.coli K12s, then was cloned into cloning vector pBluescript SK. Sequence analysis shows that the cai E gene is 612 bp long, with 10 nucletides and 6 deduced amino acids different from that reported. The gene was ligated into different vectors to construct expression vectors with different duplicons and different promoters pET CaiE, pWSK CaiE and pACYC CaiE. Then these expression vectors were transformed into E.coli BL21(DE3), after induction with 1mmol/L IPTG, CaiE was highly expressed. SDS PAGE showed the molecular weight of CaiE was 26ku, and the proportion of expression product in the total bacteria protein was pET CaiE 30%, pACYC CaiE 8%, pSC CaiE 5%, respectively.