RHODOPSIN KINASE - EXPRESSION IN BACULOVIRUS-INFECTED INSECT CELLS, AND CHARACTERIZATION OF POSTTRANSLATIONAL MODIFICATIONS

Structure-function studies of rhodopsin kinase (RK; EC 2.7.1.125) requ ire a variety of mutants, Therefore, there is need for a suitable syst em for the expression of RK mutant genes, Here we report on a study of expression of the RK gene in baculovirus-infected Sf21 cells and char acterization of the enzyme produced as purified to near homogeneity, P articular attention has been paid to the posttranslational modificatio ns, autophosphorylation and isoprenylation, found in the native bovine RK, The protein produced has been purified using, successively, hepar in-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatogra phy) and was obtained in amounts of about 2 mg from 1 liter of cell cu lture, The enzyme from the last step of purification was obtained in t wo main fractions that differ in the level of phosphorylation, The pro tein peak eluted first carries two phosphate groups per protein, where as the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C -5, C-10, C-15, and C-20 isoprenyl moieties, From these results, we co nclude that the above expression system is suitable for some but not a ll aspects of structure-function studies.