Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human β2-adrenergic receptor produced in the methylotrophic yeast Pichia pastoris

Over the last few years, Pichia pastoris has been developed into a powerful expression system for a multitude of foreign genes. Here, we demonstrate that the P. pastoris expression system has similar power to the baculovirus expression system in high-level production of twoG-protein-coupled receptors, themouse 5HT &A 5-hydroxtryptamine receptor and the human β # -adrenergic receptor. Different expression plasmids were constructed in which the cDNAs of the two receptors were cloned under the transcriptional control of the highly inducible promoter of the P. pastoris alcohol oxidase 1 (AOX1) gene. In three expression plasmids, the receptorswere fused to theSaccharomyces cereŠisiae α-factor prepropeptide and also to the c-myc tag or the FLAG tag to permit immunological detection of the receptors. After transformation into P. pastoris strains KM71 and SMD 1163, recombinant clones were selected and tested for the production of the 5HT &A receptor and the β # -adrenergic receptor by radioligand binding using [N-methyl-$H]lysergic acid diethylamide and [5,7-$H](®)CGP-12177 respectively. The production level of the 5HT &A receptor was improved by a factor of three by fusion with the α-factor prepropeptide. Also, the higher gene dosage resulting from multiple insertions of the expression cassette led to an improvement in production by a factor of two for both receptors. The addition of the adrenergic antagonist alprenolol