The human ETB endothelin receptor heterologously produced in the methylotrophic yeast Pichia pastoris shows high-affinity binding and induction of stacked membranes

The human endothelin B receptor (ETB receptor) was produced in the methylot rophic yeast Pichia pastoris is under transcriptional control of the highly inducible alcohol oxidase: 1 (AOX1) gene promoter. In the expression plasm ids pPIC9KFlagETV(B)Bio and pPIC9KFlaga Delta PET(B)Bio the ETB receptor co ding region was fused in frame to the Saccharomyces cerevisiae alpha-factor prepropeptide and the FLAG-tag. In both constructs, thr receptor was also fused to a biotinylation domain. Additionally, in Delta GPET(B)Bio the puta tive N-glycosylation site and a protease site have been deleted by site dir ected mutagenesis. Crude membranes prepared from recombinant P. pastoris re vealed specific and saturable binding of [I-125]ET-1 with a K-D of about 42 pM. Receptor levels of 60 pmol/mg and 35 pmol/mg for the Flaga Delta GPET( B)Bio and the FlagET(B)Bio construct, respectively, were determined. The ph armacological profile for ET-I. ET-2 and ET-3 were as expected for a subtyp e B endothelin (ET) receptor. Immunoblot analysis showed an apparent molecu lar mass of 55 kDa for the Kex2-processed and about 74kDa for the Kex2-unpr ocessed receptor. Contrary to the Flag Delta GPET(B)Bio construct, the Flag ET(B)Bio construct was not correctly processed by the internal Kex2 endopep tidase. As was detected by ultrastructural analysis of recombinant yeast ce lls, high-level production of the receptor resulted in the formation of sta cked membranes.