The Expression In E. Coli And Activity Detection Of Rabbit PKCε Catalytic Domain

OBJECT: We construct a prokaryotic expression vector of protein A-PKCe catalytic domain in fusion form , then express the fusion protein in E. coli and detect it's protein kinase activity. METHODS: The coding sequence of rabbit PKCs catalytic domain was subcloned into the prokaryotic expression vector pRIT5. After transforming in E. coli, the conditions of expression of the protein A-PKCε catalytic domain fusion protein was optimized and the protein A-PKCs catalytic domain fusion protein was confirmed by Western blot analysis. The pr6tein kinase activity of the fusion protein was detected by non-radioactive phospharyiation method. RESULT: The constructed recombinant expression vector protein A-PKC# catalytic domain was identified by restriction assay. The highest expression of fusion protein was gotten, the fusion protein has kinase activity after detected by non-radioactive phospharyiation method. CONCLUSION: the protein A-PKCε catalytic domain fusion protein was successfully expressed in E. coli, which lays a foundation for further studying the intracellulai signal transduction mediated by PKCε.