大肠杆菌肉碱脱水酶基因的克隆、测序及高效表达  

Cloning, Sequencing and High Expression of E. coli Carnitine Dehydratase

作　　者：范立强[1] 施惠娟[1] 贺华君[1] 袁勤生[1] 杨冠珍[2] 吴祥甫[2] 

机构地区：[1]华东理工大学生物化学研究所,上海200237 [2]中国科学院上海生物化学研究所

出　　处：《中国生物制品学杂志》2002年第3期138-142,共5页Chinese Journal of Biologicals

摘　　要：［摘 要］目的 克隆并表达大肠杆菌肉碱脱水酶基因。方法 用聚合酶链反应（PCR）从大肠杆菌K12s中扩增大肠杆菌肉碱脱水酶基因caiB，测定其DNA序列，将caiB基因重组到T7启动子控制下的表达载体pET－28a（＋）中，构建表达质粒pETCaiB，转化大肠杆菌BL21（DE3），用1mmol/L异两基硫代-β-D半乳糖苷（IPTG）诱导表达。结果 克隆得到的肉碱脱水酶编码基因 caiB长度为 1221bp，与文献报道值相比,DNA序列有26个不同，氨基酸序列只有一个不同，即302位的丙氨酸变为苏氨酸。重组菌经IPTG诱导，可高效表达，SDS－PAGE分析表达蛋白相对分子质量约43000，表达产物含量占菌体总蛋白45％以上。结论 得到了高效表达肉碱脱水酶的重组菌，为制备L-肉碱创造了条件。[Abstract] Objective To clone and express the caiB gene encoding E. coli carnitine dehydratase. Methods caiB gene was amplified from the genomic library of E. coli K12s by PCR and sequenced, then cloned into an expression vector pET- 28a( + ) under the control of promoter. The recombinant plasmid pET-CaiB was transformed to E. coli BL21(DE3) and expressed under the induction of 1 mmol/L TPTG. Results The length of the cloned caiB gene was 1221bp,and the 26 nucletides in the DNA sequence of it were different from that reported previously. However,the amino acid sequence of it was almost identical to that reported,except the change of Ala302 to Thr302.SDS - PAGE showed that the expressed product, with a relative molecular weight of 43000, contained more than 45 % of total somatic protein. Conclusion The recombinant bacterial strain for the high expression of carnitine dehydratase was constructed. It laid a foundation of preparing L - carnitine by gene engineering techniqure.

关 键 词：大肠杆菌 肉碱脱水酶 测序 高效表达 L-肉碱 基因克隆 表达 

分 类 号：Q933[生物学—微生物学] TQ925[轻工技术与工程—发酵工程]