Purification of Recombinant G Proteins from Sf9 Cells by Hexahistidine Tagging of Associated Subunits
CHARACTERIZATION OF α
AND INHIBITION OF ADENYLYL CYCLASE BY αz(*)
- From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
Abstract
A method is described for purification of G protein α and β
subunits from Sf9 cells infected with recombinant baculoviruses. The subunit to be purified is coexpressed with an associated
subunit bearing a hexahistidine tag. After adsorption of the oligomer to a Ni
-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AlF4
. The α subunits of G
, Gq, Gz, and G
and the β1
2 subunit complex were easily and efficiently purified by this method. Results were superior to established procedures in all
cases.
Purified α
was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (k
= 0.01 min
) and a very slow k
for hydrolysis of GTP (0.1-0.2 min
). GTP
S (guanosine 5′-3-O-(thio)triphosphate)•α
does not influence the activity of several adenylyl cyclases or phospholipases. Activated αz inhibits the activity of type I and type V adenylyl cyclases. It is a somewhat more potent inhibitor of type V adenylyl cyclase
than is activated α
.
Footnotes
-
↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- G protein
-
guanine nucleotide-binding regulatory protein
- GTP
S -
guanosine 5′-(3-O-thio)triphosphate
- C
E
-
polyoxyethylene 10-lauryl ether
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonic acid
- FPLC
-
fast protein liquid chromatography
- AMF
-
30 μM AlCl3, 50 mM MgCl2, and 10 mM NaF
- Ni-NTA
-
nickel-nitrilotriacetic acid.
-
↵2 The sole exception is Thr
, which is Val in αs and α
.
-
- Received October 27, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
