18] Expression, separation, and assay of protein kinase C subspecies

Abstract

Publisher Summary This chapter describes a method for the transient expression of protein kinase C subspecies, α , β I, β II, and γ in COS-7 cells using the cDNA clones isolated from rat brain. It also describes the methods for the separation and assay of these subspecies. The expression vector designated pTB701 is employed for the construction of expression plasmids of protein kinase C. The expression vector has a single cloning site of Eco RI downstream from the Abelson murine leukemia virus long terminal repeat, simian virus 40 (SV40) origin of DNA replication, and SV40 early promoter regions. The whole insert of the protein kinase C cDNA clones is incorporated into the Eco RI site of the pTB701. COS-7 cells are transfected by the calcium phosphate coprecipitation technique with glycerol shock. After a transfection period of 3.5 hr, the cells are shocked with glycerol for three min at room temperature, and fresh medium is added to each plate. Protein kinase C expressed in COS-7 cells is separated into each subspecies by hydroxyapatite column chromatography using a fast protein liquid chromatography (FPLC) system. To obtain good resolution by the hydroxyapatite column, it is necessary to partially purify the protein kinase C from the COS-7 cell extracts before this column chromatography.