Optimization of the Production of a Honeybee Odorant-Binding Protein byPichia pastoris - INRAE - Institut national de recherche pour l’agriculture, l’alimentation et l’environnement
Journal Articles Protein Expression and Purification Year : 1999

Optimization of the Production of a Honeybee Odorant-Binding Protein byPichia pastoris

Abstract

A honeybee putative general odorant-binding protein ASP2 has been expressed in the methylotrophic yeast Pichia pastoris. It was secreted into the buffered minimal medium using either the ␣-factor preprosequence with and without the Glu-Ala-Glu-Ala spacer peptide of Saccharomyces cerevisiae or its native signal peptide. Whereas ASP2 secreted using the ␣-factor preprosequence with the spacer peptide showed N-terminal heterogeneity, the recombinant protein using the two other secretion peptides was correctly processed. Mass spectrometry showed that the protein secreted using the natural peptide sequence had a mass of 13,695.1 Da, in perfect agreement with the measured molecular mass of the native protein. These data showed a native-like processing and the three disulfide bridges formation confirmed by sulfhydryl titration analysis. After dialysis, the recombinant protein was purified 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 leader sequence for secretion with correct processing in P. pastoris. The overproduction of recombinant ASP2 should allow ligand binding and mutational analysis to understand the relationships between structure and biological function of the protein.
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hal-04186829 , version 1 (24-08-2023)

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Loïc Briand, Valérie Perez, Jean-Claude Huet, Emmanuelle Danty-Berger, Claudine Masson, et al.. Optimization of the Production of a Honeybee Odorant-Binding Protein byPichia pastoris. Protein Expression and Purification, 1999, 15 (3), pp.362-369. ⟨10.1006/prep.1998.1027⟩. ⟨hal-04186829⟩

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