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 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.
Briand
L
L
Unité de Recherches de Biochimie et Structure des Protéines, INRA UR 477, Domaine de Vilvert, Jouy-en-Josas Cedex, F-78352, France.
Perez
V
V
Huet
J C
JC
Danty
E
E
Masson
C
C
Pernollet
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000235:genetics / Q000502:physiology*; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D005822:Genetic Vectors; D000072235:Mating Factor; D010455:Peptides / Q000235:genetics; D010843:Pichia; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D018035:Receptors, Odorant / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D017931:DNA Primers; D010455:Peptides; D021382:Protein Sorting Signals; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C045648:odorant-binding protein; D000072235:Mating Factor