Optimization of the production of a honeybee odorant binding protein by Pichia pastoris

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buff ered minimal medium using either the alpha-factor preprosequence with and w ithout the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or it s native signal peptide. Whereas ASP2 secreted using the alpha-factor prepr osequence with the spacer peptide showed N-terminal heterogeneity, the reco mbinant protein using the two other secretion peptides was correctly proces sed. Mass spectrometry showed that the protein secreted using the natural p eptide sequence had a mass of 13,695.1 Da, in perfect agreement with the me asured molecular mass of the native protein. These data showed a native-lik e processing and the three disulfide bridges formation confirmed by sulfhyd ryl titration analysis. After dialysis, the recombinant protein was purifie d by one-step anion exchange chromatography in a highly pure form The final expression yield after 7-day fermentation was approximately 150 mg/liter. To our knowledge, this is the first report of the use of a natural insect l eader sequence for secretion with correct processing in P. pastoris. The ov erproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein. (C) 1999 Academic Press.