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    范立强, 袁勤生, 吴祥甫. 大肠杆菌caiE基因的克隆与高效表达[J]. 华东理工大学学报(自然科学版), 2002, (2): 149-152.
    引用本文: 范立强, 袁勤生, 吴祥甫. 大肠杆菌caiE基因的克隆与高效表达[J]. 华东理工大学学报(自然科学版), 2002, (2): 149-152.
    FAN Li qing 1, YUAN Qin sheng 1*, WU Xiang fu 2. Cloning and Expression of Escherichia coli caiE Gene[J]. Journal of East China University of Science and Technology, 2002, (2): 149-152.
    Citation: FAN Li qing 1, YUAN Qin sheng 1*, WU Xiang fu 2. Cloning and Expression of Escherichia coli caiE Gene[J]. Journal of East China University of Science and Technology, 2002, (2): 149-152.

    大肠杆菌caiE基因的克隆与高效表达

    Cloning and Expression of Escherichia coli caiE Gene

    • 摘要: 用聚合酶链反应(PCR)从大肠杆菌K12s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pBluescripy SK中,测序表明:该基因有612bp,与文献报道相比,有10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ColE1为复制子,T7为启动子控制下的分泌型表达载体pET-22b( )中,构建表达质粒pETCaiE,重组到载体pACYC184中构建以p15A为复制子,Lac为启动子的表达质粒pACYC-CaiE;重组到载体pWSK129中,构建以pSC101为复制子,T7为启动子的表达质粒pSC-CaiE。上述表达质粒转化大肠杆菌BL21(DE3),经1mmol/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26ku,表达量占菌体总蛋白质的比例分别为pETCaiE30%,pACYC-CaiE8%,pSC-CaiE5%。

       

      Abstract: 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.

       

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