大肠杆菌guaC基因的克隆及高效表达

Cloning and Expression of the Gene Encoding the GMP Reductase of Escherichia coli MC4100

摘要: 以E.coli MC4100染色体DNA为模板使用PCR扩增技术克隆了鸟苷酸还原酶基因guaC，该基因全长1044bp，编码相对分子质量为37ku的蛋白质。将该基因直接克隆于PET—16b质粒的T7启动于下游，得到质粒pEXP1。转化E．coli BL21(DE3)，经异丙基硫代半乳糖苷(IPTG)诱导，获得了高效的表达。含表达质粒的菌体胞内粗提液经酶活分析表明，鸟苷酸还原酶的活性为不含质粒的宿主菌的80-85倍。

Abstract: The gene encoding GMP reductase was cloned from E. coli MC4100 by polymerase chain reaction (PCR). This gene comprises 1 044 bp, and it encodes a polypeptide of M r 37 437. The gene was inserted into the downstream of the T7 promoter of plasmid pET 16b and recombinant pEXP1 was obtained, which was then transformed into E. coli BL21(DE3). There was overexpression under the induction by IPTG. Enzyme assay showed that the activity of GMP reductase increased by 80~85 fold in the recombination strain.