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

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• 作者：Valé ; rie Sarramegna ; Isabelle Muller ; Guillaume Mousseau ; Carine Froment ; Bernard Monsarrat ; Alain Milon and Franck Talmont
• 关键词：GPCR ; Solubilization ; Detergent ; Purification ; GFP ; Mu-opioid receptor ; Pichia pastoris
• 刊名：Protein Expression and Purification
• 出版年：2005
• 出版时间：2005
• 年：2005
• 卷：43
• 期：2
• 页码：85-93
• 全文大小：321 K

文摘

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.