Solubilization, purification, and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris.
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.
Sarramegna
Valérie
V
Institut de Pharmacologie et de Biologie Structurale, UMR 5089, 205, route de Narbonne, 31077 Toulouse, Cedex 4, France.
Muller
Isabelle
I
Mousseau
Guillaume
G
Froment
Carine
C
Monsarrat
Bernard
B
Milon
Alain
A
Talmont
Franck
F
D002846:Chromatography, Affinity / Q000379:methods; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D010843:Pichia / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins