Production of the human D2S receptor in the methylotrophic yeast P. pastoris.
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 alpha-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 alpha-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 D(2) 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.
Grünewald
Sylvia
S
Max-Planck-Institut für Biophysik, Abt. Molekulare Membranbiologie, Frankfurt, Germany. gruenewald@axaron.com
Haase
Winfried
W
Molsberger
Eva
E
Michel
Hartmut
H
Reiländer
Helmut
H
D003001:Cloning, Molecular; D018628:Gene Dosage; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D017448:Receptors, Dopamine D2 / Q000235:genetics* / Q000378:metabolism
D008024:Ligands; D017448:Receptors, Dopamine D2; C113776:dopamine D2L receptor