兔PKCε催化功能域在大肠杆菌中表达与部分纯化

Expression and Partial Purification of the Catalytic Domain of Protein Kinase Cε(PKCε)in E.coli

目的通过构建蛋白激酶Cε原核表达载体以获得纯化的PKCε蛋白。方法将PKCε催化功能域(野生型和突变型)构建至原核表达载体pUC18上形成重组质粒pUC-CK和pUC-CR,将其转化至大肠杆菌DH5α细胞中诱导其高效表达。结果表达产物的SDS-PAGE电泳及Westernblot检测表明,催化功能域在大肠杆菌中成功表达,并对其进行了部分纯化。结论获得纯化的PKCε蛋白,为今后大量表达、纯化该酶蛋白作了前期铺垫。

Objective To obtain recombination PKCεprotein by expression of PKCεin E.coli DH5α. Methods The prokaryote expression vectors of PKCεwild type and mutated catalytic domain and pUC18 were constructed and transfected into E.coli DH5αcell for high expression. Results SDS PAGE and Western blotting show the expression protein was obtained. The partial purification of the PKCεCatalytic Domain were performed with His Bond Ni affinity Resin. Conclusion Our studies established the methods to obtain protein of the PKCεCatalytic Domain expressed in E.coli for further research.