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

Mariko Kato Hayashi, Tatsuya Haga

研究成果: Article

32 引用 (Scopus)

抜粋

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.

元の言語English
ページ(範囲)1232-1238
ページ数7
ジャーナルJournal of biochemistry
120
発行部数6
DOI
出版物ステータスPublished - 1996 12

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

フィンガープリント Purification and functional reconstitution with GTP-binding regulatory proteins of hexahistidine-tagged muscarinic acetylcholine receptors (m2 subtype)' の研究トピックを掘り下げます。これらはともに一意のフィンガープリントを構成します。

  • これを引用