大肠杆菌磷酸烯醇式丙酮酸羧化酶的克隆和表达

为了更好地研究磷酸烯醇式丙酮酸羧化酶的结构和功能,采用PCR(聚合酶链式反应)、双酶切和细胞转化等基因工程方法对磷酸烯醇式丙酮酸羧化酶基因进行克隆并使用SDS-聚丙烯酰胺凝胶电泳进行表达分析,扩增出一个新的大肠杆菌磷酸烯醇式丙酮酸羧化酶基因,将其基因克隆到原核表达载体pET-30a上,并导入到大肠杆菌表达菌株BL21(DE3)中。通过IPTG诱导成功表达出大肠杆菌的磷酸烯醇式丙酮酸羧化酶。所提出的方法能够高效表达磷酸烯醇式丙酮酸羧化酶,为进一步大规模表达纯化和应用提供参考。

Cloning and expression of phosphoenolpyruvate carboxykinase from Escherichia coli

In order to better study the structure and function of phosphoenolpyruvate carboxylase, the phosphoenolpyruvate carboxylase gene is cloned and expressed by PCR (polymerase chain reaction), double enzyme digestion and cell transformation. The results show that the new phosphoenolpyruvate carboxylase gene of Escherichia coli is successfully amplified and cloned into the prokaryotic expression vector pET-30a, and then introduced into Escherichia coli BL21 expression system. The phosphoenolpyruvate carboxylase of Escherichia coli is successfully induced by IPTG. The phosphoenolpyruvate carboxylase can be efficiently expressed by using the proposed method, which provides preparation for further scale expression, purification and application.