Sarramegna Valérie, Muller Isabelle, Mousseau Guillaume, Froment Carine, Monsarrat Bernard, Milon Alain, Talmont Franck
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, 205, route de Narbonne, 31077 Toulouse, Cedex 4, France.
Protein Expr Purif. 2005 Oct;43(2):85-93. doi: 10.1016/j.pep.2005.05.007.
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.
人μ-阿片受体在毕赤酵母中表达，N端有无绿色荧光蛋白（EGFP）。在摇瓶中，重组蛋白的表达产量达到每升培养基数十毫克受体。使用特异性配体的药理学研究表明，HuMOR-c-myc-组氨酸标签构建体具有典型的阿片样特征，而GFP-HuMOR-c-myc-组氨酸标签受体无法结合阿片类药物。带有六组氨酸表位标签的受体通过固定化镍亲和层析进行纯化。通过蛋白质印迹和质谱分析确认了纯化的μ-阿片受体蛋白的身份。总之，本文所述的表达、溶解和纯化策略能够分离出非常大量的纯化受体，高达12 mg/L。
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