Rhodopsin kinase: Expression in baculovirus-infected insect cells, and characterization of post-translational modifications* (G-protein-coupled receptor kinasesyrod outer segmentyautophosphorylationyisoprenylationySpodoptera frugiperda cells)

Structure–function studies of rhodopsin kinase (RK; EC 2.7.1.125) require a variety of mutants. Therefore, there is need for a suitable system 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 characterization of the enzyme produced as purified to near homogeneity. Particular attention has been paid to the posttranslational modifications, autophosphorylation and isoprenylation, found in the native bovine RK. The protein produced has been purified using, successively, heparin-Sepharose, Mono Q, and Mono S FPLC (fast protein liquid chromatography) and was obtained in amounts of about 2 mg from 1 liter of cell culture. The enzyme from the last step of purification was obtained in two main fractions that differ in the level of phosphorylation. The protein peak eluted first carries two phosphate groups per protein, whereas the second protein peak is monophosphorylated. Further, while both peaks are isoprenylated, the isoprenyl groups consist of mixtures of C5, C10, C15, and C20 isoprenyl moieties. From these results, we conclude that the above expression system is suitable for some but not all aspects of structure–function studies. Rhodopsin kinase (RK; EC 2.7.1.125), specific to the mammalian retina, phosphorylates rhodopsin and thus initiates the desensitization cascade (2, 3). RK, a protein of about 62 kDa molecular mass, has been studied extensively, and methods are available for its purification from bovine rod cell outer segment (ROS) (4, 5). We are interested in structure–function studies of this enzyme that require preparation of a variety of mutants (6). These, in turn, require expression of the RK mutant genes in a suitable system and a method for purification in adequate amounts. In this paper, we report on expression of the RK gene in baculovirus-infected Sf21 insect cells and purification and characterization of the produced protein. The baculovirus-infected insect cell system has been used for the production of a large variety of proteins. In particular, several G-protein-coupled receptor kinases have been produced by using this system (7–9). In the present work, particular attention has been paid to characterization of posttranslational modifications of the protein as produced in the insect cells, specifically, isoprenylation and autophosphorylation that have been found in RK from bovine ROS (10, 11). We find that RK is produced in satisfactory amounts. On purification it distributes into two main fractions that differ from each other in regard to having either one or two phosphate groups per protein. Both fractions are isoprenylated; however, there is heterogeneity in the isoprenyl groups, these being mixtures of C5–C20 chain lengths. RK from the insect cells [RK(BV)] is indistinguishable from RK as prepared from bovine ROS [RK(ROS)] in regard to specific activity, temperature sensitivity, pH optimum, and mobility in SDSyPAGE. We conclude that the above expression system promises to be useful in some but not all structure–function studies of the enzyme. MATERIALS AND METHODS