Purification and Functional Reconstitution with GTP-Binding Regulatory Proteins of Hexahistidine-Tagged Muscarinic Acetyicholine Receptors (m2 Subtype)
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- Hayashi Mariko Kato
- Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo
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- Haga Tatsuya
- Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, The University of Tokyo
書誌事項
- タイトル別名
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- Purification and Functional Reconstitution with GTP-Binding Regulatory Proteins of Hexahistidine-Tagged Muscarinic Acetylcholine Receptors (m2 Subtype).
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説明
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.
収録刊行物
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- The Journal of Biochemistry
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The Journal of Biochemistry 120 (6), 1232-1238, 1996
社団法人 日本生化学会
