Graber S G, Figler R A, Garrison J C
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
J Biol Chem. 1992 Jan 15;267(2):1271-8.
The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S.
利用杆状病毒表达系统，异三聚体鸟嘌呤核苷酸结合蛋白Gi1、Gi2、Gi3、G0和Gs的α亚基已在草地贪夜蛾细胞（Sf9）中过表达。Gi1α、Gi2α、Gi3α和G0α已从感染的草地贪夜蛾（SF9）细胞中纯化至同质并进行了特性鉴定。从300毫升感染细胞培养物中已获得高达1.8毫克的纯化重组Gα。重组α亚基被肉豆蔻酰化，并且仅在存在βγ亚基的情况下被百日咳毒素ADP核糖基化。它们以低纳摩尔解离常数和0.8摩尔/摩尔或更高的化学计量比结合鸟苷5'-3-O-（硫代）三磷酸（GTPγS）。基于rGi1α、rGi2α和rGi3α能够恢复在GTPγS作用下转变为低亲和力状态的大鼠肝细胞膜中高亲和力血管紧张素II结合的能力，它们能够与血管紧张素II受体相互作用。
