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Protein Expr Purif

Title:		Optimization of the production of a honeybee odorant-binding protein by Pichia pastoris
Author(s):		Briand L; Perez V; Huet JC; Danty E; Masson C; Pernollet JC;
Address:		"Unite de Recherches de Biochimie et Structure des Proteines, INRA UR 477, Domaine de Vilvert, Jouy-en-Josas Cedex, F-78352, France"
Journal Title:		Protein Expr Purif
Year:		1999
Volume:		15
Issue:		3
Page Number:		362 - 369
DOI:		10.1006/prep.1998.1027
ISSN/ISBN:		1046-5928 (Print) 1046-5928 (Linking)
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 alpha-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 alpha-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"
Keywords:		"Amino Acid Sequence Animals Bees/genetics/*physiology Chromatography, High Pressure Liquid Chromatography, Ion Exchange Cloning, Molecular/methods DNA Primers Genetic Vectors Mating Factor Peptides/genetics Pichia Polymerase Chain Reaction Protein Sorting;"
Notes:		"MedlineBriand, L Perez, V Huet, J C Danty, E Masson, C Pernollet, J C eng 1999/03/27 Protein Expr Purif. 1999 Apr; 15(3):362-9. doi: 10.1006/prep.1998.1027"