EXPRESSION OF FUNCTIONAL-MOUSE 5-HT5A SEROTONIN RECEPTOR IN THE METHYLOTROPHIC YEAST PICHIA-PASTORIS - PHARMACOLOGICAL CHARACTERIZATION ANDLOCALIZATION

The methylotrophic yeast Pichia pastoris was tested for heterologous e xpression of the mouse 5-HT5A receptor, Three different expression pla smids were constructed where the cDNA of the receptor was cloned under the transcriptional control of the highly inducible promotor of the P . pastoris alcohol oxidase 1 (AOX1) gen, The expression plasmids diffe red with respect to the signal sequences used for N-terminal fusion, I n two cases the coding region was additionally fused to the c-myc tag to permit immunological detection of the receptor. Expression of funct ional receptor after transformation of strain GS115 was detected by ra dioligand binding using [H-3]LSD. The construct with the best expressi on levels in strain GS115 was used for transformation of the protease deficient strain SMD1163, Here, the expression level was 2-8 times hig her. Whole cells as well as crude membrane preparations of recombinant clones showed saturable binding of [H-3]LSD with a K-d of approximate to 1.9 nM. Receptor concentrations of approximate to 22 pmol/mg membr ane protein revealed the potential of the P. pastoris expression syste m for high level expression of membrane proteins. The pharmacological properties were comparable to those reported for the receptor expresse d in mammalian systems.