Pichia pastoris is a methylotrophic yeast that is commonly used for its ability to express and secrete heterologous proteins. However, some proteins are not readily secreted in P pastoris and so adjustments in the secretion pathway must be made in order to achieve secretion. The Lin-Cereghino lab previously developed mutant strains using restriction enzyme-mediated integration that enabled P pastoris to secrete Pgalactosidase at higher levels than the wild type strain. This study focuses on characterizing the random pREMI-Z mutations in the genomic DNA and examining their secretory phenotype, in hopes of creating a super secretor strain. The ah3 mutant was specifically chosen and characterized for its ability to secrete HRP and SLPI proteins and the effect of the pREMI-Z mutation on the morphology of the cells using transmission electron microscopy. An examination into the AH3 protein yielded a comparative B-galactosidase secretion study between the ah3 mutant and ah3 mutant cells transformed with the pKANB-AH3 rescue construct. Lastly, a cell localization experiment was done to examine where the AH3 protein may be found. These experiments help to increase the current understanding of the secretion pathway in Pichia pastoris and serves as an outline of how to characterize other pREMI -Z mutant strains.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-1768 |
| Date | 01 January 2011 |
| Creators | Larsen, Sasha Ellen Marie |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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