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. Co²⁺-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]GTpγS to the G-protein G₁₂ reconstituted in lipid vesicles with purified m2 receptors. The extent of stimulation of [³⁵S]GTPγS 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.