Purification and functional reconstitution with GTP-binding regulatory proteins of hexahistidine-tagged muscarinic acetylcholine receptors (m2 subtype).
We have expressed human m2 muscarinic acetylcholine receptors tagged with six histidine residues at the carboxy-terminal region in insect cells (Sf9) and purified them using metal-immobilized Chelating Sepharose gels. Co(2+)-immobilized gels were found to be much more efficient for purification of m2 receptors than gels containing Ni2+ or other metal ions. Twenty-fold purification was attained by a simple, single-step procedure, and approximately 40% of solubilized receptors were recovered as a partially purified preparation with a specific activity of 1.6 nmol/mg of protein. Purified receptors were functionally active in that carbamylcholine stimulated binding of [35S]GTP gamma S to the G-protein G12 reconstituted in lipid vesicles with purified m2 receptors. The extent of stimulation of [35S] GTP gamma S binding to G12 by hexahistidine-tagged m2 receptors was essentially the same as that observed for m2 receptors that lack histidine tags. In addition, palmitoylation at the carboxy-terminal region was not impaired by the hexahistidine-tag fusion. The method described in this study should be applicable to the purification of other G-protein-coupled receptors in functionally active form.
Hayashi
M K
MK
Department of Biochemistry, Faculty of Medicine, University of Tokyo.
Haga
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003300:Copper / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006601:Hexoses; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism*; D018411:Spodoptera
D006601:Hexoses; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D003300:Copper; D009532:Nickel; D019204:GTP-Binding Proteins