Rhodopsin kinase: Expression in mammalian cells and a two-step purification

A suitable system for expression of the rhodopsin kinase (RK) gene and its mutants is needed for structure-function studies of RK. Previously, investigation of the baculovirus system showed satisfactory production of RK, but posttranslational isoprenylation was deficient, We now report on a comparative study of expression of the RK gene in yeast (Pichia pastoris), COS-1 cells and in an HEK293 stable cell line, Expression in COS-1 cells. by using pCMV5 vector, is the most satisfactory. A two-step procedure for purification of the expressed enzyme with an N-terminal histidine tag has been developed, The purified enzyme has correct posttranslational modifications and shows a somewhat broader pH vs. catalytic activity profile than the wild-type enzyme.