Datensatz

Zusammenfassung

In order to evaluate the methylotrophic yeast Pichia pastoris as means for high-yield production of homogenous D_2S receptor protein, we have expressed the unmodified D_2S receptor and various D_2S receptor fusion constructs under the transcriptional control of the highly inducible promotor of the P. pastoris alcoholoxidase 1 gene in strain SMD1163. Fusion of the D_2S receptor gene to the α-factor preprosequence proved to be essential for receptor production. For the receptor fusion constructs a gene dosage of more than two copies per cell increased production levels three- to sixfold. Adding various dopaminergic ligands to the induction medium increased yields up to tenfold, reaching 51,500 ± 5700 receptors/cell. Immunoblot analysis of the effect of tunicamycin on D_2S receptor fusion proteins and immunoprecipitation of metabolically labeled wild-type and glycosylation-deficient D_2S receptor fusion proteins revealed that the high-mannose-type glycosylation of the D_2S receptor prevents cleavage of the α-factor prosequence by the Kex2 endopeptidase. Abolishing glycosylation restored correct processing. Immunogold electron microscopy showed that recombinant yeast cells overproducing the D_2S receptor developed membrane stacks harboring the receptor protein. The pharmacological profile of the recombinant D_2S receptor was similar to that reported for neuronal D₂ receptors independent of glycosylation and processing. In conclusion, the D_2S receptor can readily be produced in P. pastoris with high yield suitable for receptor purification and future structural studies.