Expression of Gsα in Escherichia coli: Purification and properties of two forms of the protein

摘要: Cloning of complementary DNAs that encode either two forms the α subunit guanine nucleotidebinding regulatory protein (Gs) stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M. P., Casey, P. J., and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375–11381). A rapid procedure for purification milligram quantities is described. As expressed E. coli, both Gsα, (apparent molecular weights 45,000 52,000) bind guanosine 5′-(3-O-thio)triphosphate stoichiometrically. The also hydrolyze GTP, although at different rates (i.e. 0.13•min−1 0.34•min−1 20 °C 45- 52-kDa forms, respectively). These reflect differences rate dissociation GDP from proteins. Both recombinant have essentially same kcat GTP hydrolysis, ∼4. min−1. Recombinant interacts functionally with G βγ subunits β-adrenergic receptors. can ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires addition subunits. reconstitute GTP-, isoproterenol + 5′-(3-O-thio)triphosphate-, fluoride-stimulated activity S49 cyc− membranes maximal levels, their specific activities this are lower than observed Gs purified rabbit liver. Experiments bovine brain indicate affinity Gsα 5–10 times liver under assay conditions; however, intrinsic capacity activate normal. findings suggest when may fail undergo a posttranslational modification crucial high interaction cyclase.