title: Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris.
abstract: The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Pachai S
authors.initials: PS
authors.lastname: Moua
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Gonzalez
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Kristin T
authors.initials: KT
authors.lastname: Oshiro
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Vivian
authors.initials: V
authors.lastname: Tam
authors.affiliation: Department of Chemistry, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Zhiguo Harry
authors.initials: ZH
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Chang
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Wilson
authors.initials: W
authors.lastname: Leung
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Amy
authors.initials: A
authors.lastname: Yon
authors.affiliation: Department of Biomedical Sciences, Arthur A. Dugoni School of Dentistry, University of the Pacific, San Francisco, CA 94103, United States.
authors.forename: Der
authors.initials: D
authors.lastname: Thor
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Sri
authors.initials: S
authors.lastname: Venkatram
authors.affiliation: Department of Chemistry, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Franz
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Risser
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States.
authors.forename: Joan
authors.initials: J
authors.lastname: Lin-Cereghino
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, United States. Electronic address: glincere@pacific.edu.
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Lin-Cereghino
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Fluorescence; Fusion protein; Maltose binding protein; Pichia pastoris; Proteolysis; Recombinant expression; Secretion