Purification and functional reconstitution with GTP-binding regulatory proteins of hexahistidine-tagged muscarinic acetylcholine receptors (m2 subtype)

Mariko Kato Hayashi, Tatsuya Haga

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Abstract

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.

Original languageEnglish
Pages (from-to)1232-1238
Number of pages7
JournalJournal of biochemistry
Volume120
Issue number6
DOIs
Publication statusPublished - 1996 Dec

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Keywords

  • G-protein
  • Histidine-tag
  • Muscarinic receptor
  • Purification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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