人源细胞色素C在大肠杆菌中的重组表达和分离纯化

Expression and Purification of Recombinant Human Cytochrome C in Escherichia coli

细胞色素C在电子传递、缺氧应激和细胞凋亡的过程中起重要作用。通过人源细胞色素C和酵母细胞色素C亚铁血红素裂解酶共表达,从大肠杆菌中获得了可溶性的重组人源细胞色素C蛋白。细菌经过超声破碎后,获得的上清经350g/L硫酸铵沉淀去除杂蛋白,再经过两次SP-Sepharose Fast Flow阳离子交换层析,获得了纯度大约为80%的人源细胞色素C。每升菌可产出10mg以上重组细胞色素C。人源细胞色素C的重组表达和纯化有助于细胞色素C功能的深入研究和应用。

Cytochrome C plays important roles in electron transferring, oxidative stress and apoptosis. In this study,soluble cytochrome C was accumulated in cytoplasm of E. coli by utilizing the co-expression of human cytochrome c and yeast heine lyase from a single plasmid. After ultrasonic disruption of the bacteria, a lot of contaminated proteins were discarded by addition of 350 g/L ammonium sulfate into the supernatant. Then the recombinant human cytochrome C was purified to 80% homogeneity after two times cation exchange chromatography on SP-Sepharose Fast Flow. Yields of cytochrome C greater than 10 mg per liter culture were attained. This efficient system for producing human cytochrome C is helpful for us to understand the roles of this protein in biological processes and therapy of human diseases relevant to apoptosis and oxidative stress.