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.
Bruel
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Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Cha
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Reeves
P J
PJ
Getmanova
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E
Khorana
H G
HG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000201:enzymology
D005136:Eye Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; C496764:GRK1 protein, human