不相容双质粒共表达大肠杆菌肉碱脱水酶基因caiB及其辅因子合成酶基因caiE

Coexpression of caiB and caiE with Incompatible Plasmids in E.coli

基因 cai B和基因 cai E分别编码肉碱脱水酶及其辅因子合成酶 ,携带这两个基因的重组菌可以共表达两个外源蛋白 ,得到高活性肉碱脱水酶。分别构建这两个基因的表达质粒 p ET2 8cai B和 p ET2 2 cai E,利用双抗生素筛选法 ,获得能稳定遗传的双质粒转化子。经 IPTG诱导 ,两个基因共表达 ,表达量分别占菌体总蛋白的 39%和 2 0 % ,共转化菌的酶活力比 p ET2 8cai B单转化菌提高了 2 .3倍 。

A recombinant bacterium exhibiting high enzymatic activity could be obtained by cloning and coexpression of both caiB and caiE genes in a host of E.coli BL21(DE3), which encode carnitine dehydratase and a protein that may be related to the synthesis of cofactor for carnitine dehydratase, respectively. The genes were isolated from pSK-caiB or pSK-caiE and then were ligated into expression vector pET28a(+) or pET22b(+) to construct plasmid pET28caiB or pET22caiE. These two plasmids can be stably transformed and maintained in E.coli BL21(DE3) when both ampicillin and kanamycin were presented in the selective medium. After induction with IPTG, both caiB and caiE genes were coexpressed and the expressed products accounted for 39% and 20% of the total proteins in the host. Compared with E.coli BL21(DE3) containing only pET28caiB, the activity of carnitine dehydratase increased 2.3-fold in the same strain containing two plasmids. A new method for coexpression of proteins in E.coli containing two incompatible plasmids in which two different antibiotic resistant markers were included was also established.