Purification of heterotrimeric G protein alpha subunits by GST-Ric-8 association: primary characterization of purified G alpha(olf).
Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gα(olf) for the first time and unprecedented yield of Gα(q) and Gα(13). Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF(4)(-) (GTP mimicry) and found to be >80% pure, bind guanosine 5'-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gα(olf) showed that it binds GTPγS at a rate marginally slower than Gα(s short) and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gα(s short).
Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.
PuiYee
P
Chan
Meital
M
Gabay
Forrest A
FA
Wright
Wei
W
Kan
Sukru S
SS
Oner
Stephen M
SM
Lanier
Alan V
AV
Smrcka
Joe B
JB
Blumer
Gregory G
GG
Tall
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D004789:Enzyme Activation; D044385:GTP-Binding Protein alpha Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera
D044385:GTP-Binding Protein alpha Subunits; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases