Expression and pharmacological characterization of the human mu-opioid receptor in the methylotrophic yeast Pichia pastoris.
The human mu-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae alpha-mating factor signal sequence has been expressed in the methylotrophic yeast Pichia pastoris using the host promoter of the alcohol oxidase-1 gene. Cell membranes exhibited specific and saturable binding of the opioid antagonist [3H]diprenorphine (Kd = 0.2 nM and Bmax = 400 fmol/mg protein or 800 sites/cell). Competition studies with non-selective, and mu-, delta- and kappa-selective opioid agonists and antagonists revealed a typical mu-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.
Talmont
F
F
Institut de Pharmacologie et de Biologie Structurale, CNRS-UPR 9062, Toulouse Cedex, France.
Sidobre
S
S
Demange
P
P
Milon
A
A
Emorine
L J
LJ
D002462:Cell Membrane; D004174:Diprenorphine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015870:Gene Expression; D006801:Humans; D009292:Narcotic Antagonists / Q000378:metabolism; D009294:Narcotics / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D009292:Narcotic Antagonists; D009294:Narcotics; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D004174:Diprenorphine