Rhodopsin kinase: expression in baculovirus-infected insect cells, and characterization of post-translational modifications.
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 post-translational 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.
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Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Bruel
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Inglese
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Khorana
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HG
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013696:Temperature
D005136:Eye Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1