Expression, Purification And Activity Analysis Of The Catalytic Domain Of Rabbit Protein Kinase C ε (PKCε)

Objective: In this study, the prokaryotic expression plasmids of fusion protein of protein A-PKC ε catalytic domain (wild type and dominant negative type) are respectively constructed. The fusion proteins are expressed in E.coli Stable 2 cell transformed with the recombinant plasmids. The protein kinase activity of the expressed fusion proteins is analysed.Methods: The prokaryotic high-level expression vector (pPA) of protein A is constructed. Then the coding sequence of rabbit PKC ε catalytic domain (wild type and dominant negative type) is respectively subcloned into the pPA, to form the recombinant plasmids pPA-FLC and pPA-ANC, which are expressed in E.coli Stable2 cell. The expressed fusion proteins are confirmed by Western blotting. And the activity of expressed protein A fusion proteins is detected by non-radioactive protein kinase assay system.Results: The recombinant expression vector pPA and recombinant plasmids pPA-FLC, pPA-ANC have confirmed by restriction enzyme assay and sequencing. The fusion proteins are expressed in E.coli Stable 2 cell transformed with the recombinant plasmids. And the fusion proteins have shown protein kinase activity. Conclusions: The active fusion protein of protein A-PKC ε catalytic domain has successfully expressed in E.coli. The fusion proteins have purified by IgG-Sepharose and will be of benefit to searching the direct substrate of PKC ε in rabbit cardiomyocyte.