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

Mariko Kato Hayashi, Tatsuya Haga

Research output: Contribution to journalArticle

32 Citations (Scopus)

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
Publication statusPublished - 1996 Dec
Externally publishedYes

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His-His-His-His-His-His
Muscarinic M2 Receptors
Muscarinic Receptors
Guanosine Triphosphate
GTP-Binding Proteins
Purification
Gels
Histidine
Metals
Lipoylation
Sf9 Cells
Proteins
Carbachol
G-Protein-Coupled Receptors
Chelation
Sepharose
Metal ions
Insects
Fusion reactions
Ions

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and functional reconstitution with GTP-binding regulatory proteins of hexahistidine-tagged muscarinic acetylcholine receptors (m2 subtype). / Hayashi, Mariko Kato; Haga, Tatsuya.

In: Journal of Biochemistry, Vol. 120, No. 6, 12.1996, p. 1232-1238.

Research output: Contribution to journalArticle

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