Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris
[摘要] The human μ-opioid receptor cDNA from which the 32 amino-terminal codons were substituted by the Saccharomyces cerevisiae α-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 (K d = 0.2 nM and B max = 400 fmol/mg protein or 800 sites/cell). Competition studies with non-selective, and μ-, δ- and κ-selective opioid agonists and antagonists revealed a typical μ-opioid receptor binding profile, suggesting proper folding of the protein in yeast membranes.
[发布日期] [发布机构]
[效力级别] [学科分类] 生物化学/生物物理
[关键词] μ-Opioid receptor;Heterologous expression;Pichia pastoris;Membrane receptor;Methylotrophic yeast;HuMOR;human μ-opioid receptor;AOX1;gene for alcohol oxidase-1;STE2;gene for α-mating factor receptor;MD;minimal dextrose;MM;minimal methanol;BMGY;buffered minimal glycerol-complex medium;BMMY;buffered minimal methanol-complex medium;PMSF;phenylmethylsulfonyl fluoride;5-HT;5-hydroxytryptamine;DPN;diprenorphine;DAGO;[d-Ala2;N-Me-Phe4;Gly-ol5]enkephalin;DTLET;Tyr-d-Thr-Gly-Phe-Leu-Thr;U-50;488;trans-(−)-3;4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzene acetamide;β-FNA;β-funaltrexamine;ICI-174;846;N;N-diallyl-Tyr-Aib-Aib-Phe-Leu;NorBNI;nor-binaltorphimine [时效性]
