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α-Factor Prepropeptide; Cellular Localization; Heterologous Production; Human Dopamine D2S Receptor; Immunogold Electron Microscopy; Membrane Protein; P. pastoris; Processing
Abstract:
In order to evaluate the methylotrophic yeast Pichia pastoris as means for high-yield production of homogenous D2S receptor protein, we have expressed the unmodified D2S receptor and various D2S 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 D2S 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 D2S receptor fusion proteins and immunoprecipitation of metabolically labeled wild-type and glycosylation-deficient D2S receptor fusion proteins revealed that the high-mannose-type glycosylation of the D2S 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 D2S receptor developed membrane stacks harboring the receptor protein. The pharmacological profile of the recombinant D2S receptor was similar to that reported for neuronal D2 receptors independent of glycosylation and processing. In conclusion, the D2S receptor can readily be produced in P. pastoris with high yield suitable for receptor purification and future structural studies.
