大肠杆菌CaiDE的克隆测序及表达研究

Cloning and Expressing of E.coli CaiDE

用聚合酶链反应 (PCR)从大肠杆菌K12s基因组DNA中扩增大肠杆菌肉碱消旋酶及相关酶基因CaiDE ,将其克隆到克隆载体pBluescriptSK中 ,该基因有 15 0 0bp ,与文献报道相比 ,有 2 1个核苷酸不同 ,相应翻译的氨基酸有 11个差异。将该基因重组到corE1为复制子 ,T7为启动子控制下的表达载体pET 2 4a(+)中 ,构建表达质粒pETCaiDE ,转入大肠杆菌BL2 1(DE3)中 ,经 1mmol/L异丙基硫代 β D 半乳糖苷 (IPTG)诱导后进行SDS PAGE分析 ,表达蛋白相对分子质量为 32 0 0 0和 2 6 0 0 0 ,表达量占菌体总蛋白质的比例分别为 10 %和 5 %

The 1500bp caiDE gene, which encodes carnitine racemase and a enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities, was first isolated by PCR from a genomic library of E. coli K12s, then was ligated into plasmid pET24a(+)to construct expression vector pET CaiDE. After 3 h induction with 1mmol/L IPTG, two proteins with M r of 32000 and 26000 was expressed in E. coli BL21(DE3). The proportion of expression product in the total protein of the bacteria was 10% and 5% respectively.