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Abstract:
The human endothelin B receptor (ET(B) receptor) was produced in the methylotrophic yeast Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 (AOX1) gene promoter. In the expression plasmids pPIC9KFlagET(B)Bio and pPIC9KFlag deltaGPET(B)Bio the ET(B) receptor coding region was fused in frame to the Saccharomyces cerevisiae alpha-factor prepropeptide and the FLAG-tag. In both constructs, the receptor was also fused to a biotinylation domain. Additionally, in pPIC9KFlag deltaGPET(B)Bio the putative N-glycosylation site and a protease site have been deleted by site directed mutagenesis. Crude membranes prepared from recombinant P. pastoris revealed specific and saturable binding of [125I]ET-1 with a K(D) of about 42 pM. Receptor levels of 60 pmol/mg and 35 pmol/mg for the Flag deltaGPET(B)Bio and the FlagET(B)Bio construct, respectively, were determined. The pharmacological profile for ET-1, ET-2 and ET-3 were as expected for a subtype B endothelin (ET) receptor. Immunoblot analysis showed an apparent molecular mass of 55 kDa for the Kex2-processed and about 74 kDa for the Kex2-unprocessed receptor. Contrary to the Flag deltaGPET(B)Bio construct, the FlagET(B)Bio construct was not correctly processed by the internal Kex2 endopeptidase. As was detected by ultrastructural analysis of recombinant yeast cells, high-level production of the receptor resulted in the formation of stacked membranes.
