Expression of MBP-EGFP in betagalactosidase supersecretor (bgs) strains of Pichia pastoris
Poster Number
16
Format
Poster Presentation
Faculty Mentor Name
Geoff Lin-Cereghino
Faculty Mentor Department
Biological Sciences
Additional Faculty Mentor Name
Joan Lin-Cereghino
Additional Faculty Mentor Name
Doug Riser
Abstract/Artist Statement
Pichia pastoris is a methylotrophic yeast that can be used to express recombinant proteins. Its advantages over protein expression with Escherichia coli include the ability to secrete proteins out of the cell and the ability to perform the required post-translational modifications to the recombinant proteins. However, with certain proteins, secretion is not always efficient. To study this problem, we have been examining the secretion of fusions of the enhanced green fluorescent protein (EGFP) and the maltose binding protein (MBP). When the EGFP-MBP fusion protein is expressed in wild type strains, it is not secreted efficiently, is released as one protein, and often goes to the vacuole inside the cell. However, for the expression of MBP-EGFP in wild type strains, the protein is produced more efficiently, is released as two separate proteins (separate MBP and EGFP) and is not localized in the same areas in the cell as EGFP-MBP. In this study, we observed the localization of MBPEGFP using fluorescence microscopy and quantified the expression of the protein with western analysis in beta-galactosidase super secretor (bgs) mutant strains, comparing these results to what occurs in the wild type P. pastoris strain. Our results suggest that there may be a connection between localization and proteolysis, and proteolysis may be less frequent in certain bgs strains.
Location
DeRosa University Center, Ballroom
Start Date
25-4-2015 2:00 PM
End Date
25-4-2015 4:00 PM
Expression of MBP-EGFP in betagalactosidase supersecretor (bgs) strains of Pichia pastoris
DeRosa University Center, Ballroom
Pichia pastoris is a methylotrophic yeast that can be used to express recombinant proteins. Its advantages over protein expression with Escherichia coli include the ability to secrete proteins out of the cell and the ability to perform the required post-translational modifications to the recombinant proteins. However, with certain proteins, secretion is not always efficient. To study this problem, we have been examining the secretion of fusions of the enhanced green fluorescent protein (EGFP) and the maltose binding protein (MBP). When the EGFP-MBP fusion protein is expressed in wild type strains, it is not secreted efficiently, is released as one protein, and often goes to the vacuole inside the cell. However, for the expression of MBP-EGFP in wild type strains, the protein is produced more efficiently, is released as two separate proteins (separate MBP and EGFP) and is not localized in the same areas in the cell as EGFP-MBP. In this study, we observed the localization of MBPEGFP using fluorescence microscopy and quantified the expression of the protein with western analysis in beta-galactosidase super secretor (bgs) mutant strains, comparing these results to what occurs in the wild type P. pastoris strain. Our results suggest that there may be a connection between localization and proteolysis, and proteolysis may be less frequent in certain bgs strains.