pfl-act基因在大肠杆菌中的高效表达及其纯化

丙酮酸甲酸裂解酶是肠道细菌在厌氧代谢中十分关键的酶,丙酮酸甲酸裂解酶激活因子(pyruvateform ate lyase activator,PFL-A)在功能上具有重要的作用。为进一步研究PFL-A的激活机理,以大肠杆菌K-12的基因组为模板,通过G enB ank上公布的序列设计引物,扩增出目的基因,克隆到pM D 18-T载体,经测序,所扩增出的基因与p f l-act基因具有99%的同源性。将p f l-act连接到高效表达载体pET-22b( )中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导,结果发现,p f l-act以包涵体形式表达。改变诱导剂、诱导剂量或培养温度,对包涵体的形成均没有明显的影响。

High expression and purification of pfl-act gene in Escherichia coli

Pyruvate formate lyase(PFL) is a key enzyme acting in anaerobic metabolic pathway in enteric bacteria.As activator of PFL,pyruvate formate lyase activator(PFL-A) shows important effect in the function.For the further research in the activating mechanism,pfl-act was amplified with the genomic DNA of Escherichia coli using the primers designed by the sequence from GenBank,ligated with pMD18-T vector,and sequenced.The cloning pfl-act nucleotide had 99% identity with the objective gene.The segment of pfl-act was ligated with the high expressing vector pET-22b( ),induced by IPTG.The result showed that pfl-act was expressed by inclusion body.The inclusion body was not obviously changed by optimized the amount of inducer,the inducer or the temperature of cultivating.