pyrG基因的克隆表达及CTPS的活性测定

为提高乳清酸到三磷酸胞苷(CTp)的转化效率,克隆并表达了编码CTP合成酶(CTPS)的基因pyrG。根据Genbank的资料,设计了pyrG的引物,经PCR扩增、酶切后,将pyrG插入到表达载体pET-28a中,构建了重组质粒pET28a-pyrG,然后转化到大肠杆菌BL21(DE3)中,乳糖诱导表达后,经SDS-PAGE对表达产物进行分子量鉴定。分离纯化后,对表达产物CTPS进行活性测定,并对转化工艺进行初步研究。结果:构建的工程菌产生了一种相对分子质量在60.0k的蛋白,该蛋白显示出CTPS的活性,并且可以转化乳清酸为CTP。

pyrG Cloning and Expressing in E. coli with CTPS Activity

To increase the biotransformation efficiency from orotic acid to CTP,pyrG,a gene encoding a CTP synthetase,was cloned in E.coli and its expression was detected.pyrG was amplified from Ecoli by PCR using the primers designed according to the information from GeneBank.The PCR product was then digested and was inserted into the protein expression vector pET-28a.The recombination plasmid pET28a-pyrG was transformed into the E.coli strain BL21(DE3).After pyrG was induced to express in BL21 by lactose,a protein of molecular weight of 60.0k was identified by SDS-PAGE.The purified protein was detected with CTPS activity and was able to transform orotic acid to CTP.The genetic engineering strain pET28a/pyrG possessing the initial industrial production prospects was discussed.