A suitable system for expression of the rhodopsin kinase (RK) gene and its
mutants is needed for structure-function studies of RK. Previously, investi
gation of the baculovirus system showed satisfactory production of RK, but
posttranslational isoprenylation was deficient, We now report on a comparat
ive study of expression of the RK gene in yeast (Pichia pastoris), COS-1 ce
lls 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 purificati
on of the expressed enzyme with an N-terminal histidine tag has been develo
ped, The purified enzyme has correct posttranslational modifications and sh
ows a somewhat broader pH vs. catalytic activity profile than the wild-type
enzyme.