A novel, generic and effective method for the rapid purification of G protein-coupled receptors

被引：8

作者：

Magnin, Thierry ^{[1
]}

Fiez-Vandal, Cedric ^{[1
]}

Potier, Noelle ^{[2
]}

Coquard, Aline ^{[1
]}

Leray, Isabelle ^{[1
]}

Steffan, Tania ^{[1
]}

Logez, Christel ^{[1
]}

Alkhalfioui, Fatima ^{[1
]}

Pattus, Franc ^{[1
]}

Wagner, Renaud ^{[1
]}

机构：

[1] Inst Gilbert Laustriat, Pole API, LC1 UMR 7175, F-67412 Illkirch Graffenstaden, France

[2] CNRS, Inst Chim, LC3, UMR 7177,ISIS, F-67083 Strasbourg, France

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2009年 / 64卷 / 01期

关键词：

G protein-coupled receptor; Pichia pastoris; Solubilization; Purification; Cannabinoid; Opioid; Ligand; LARGE-SCALE PURIFICATION; MEMBRANE-PROTEIN; EXPRESSION; YEAST;

D O I：

10.1016/j.pep.2008.09.007

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed. (C) 2008 Elsevier Inc. All rights reserved.

引用

页码：1 / 7

页数：7

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