Solubilization, purification, and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris

被引：36

作者：

Sarramegna, V ^{[1
]}

Muller, I ^{[1
]}

Mousseau, G ^{[1
]}

Froment, C ^{[1
]}

Monsarrat, B ^{[1
]}

Milon, A ^{[1
]}

Talmont, F ^{[1
]}

机构：

[1] UMR 5089, Inst Pharmacol & Biol Struct, F-31077 Toulouse, France

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2005年 / 43卷 / 02期

关键词：

GPCR; solubilization; detergent; purification; GFP; mu-opioid receptor; Pichia pastoris;

D O I：

10.1016/j.pep.2005.05.007

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The human mu-opioid receptor was expressed in Pichia pastoris with or without EGFP at the N-terminal end. Expression yields of the recombinant proteins reached several tens of milligram of receptor per liter of culture medium in shacked flasks. Pharmacological studies using specific ligands demonstrated a typical opioid profile for the HuMOR-c-myc-his-tag construct, whereas the GFP-HuMOR-c-myc-his-tag receptor was unable to bind opioid drugs. The hexahistidine epitope-tagged receptors were purified by immobilized-nickel affinity chromatography. The identity of the purified mu-opioid receptor proteins was confirmed by Western blot and mass spectrometry analysis. In conclusion, the expression, solubilization, and purification strategies described herein allow to isolate very high quantities of purified receptor, up to 12 mg/L. (c) 2005 Elsevier Inc. All rights reserved.

引用

页码：85 / 93

页数：9

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