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Date of Award
2012
Document Type
Thesis - Pacific Access Restricted
Degree Name
Master of Science (M.S.)
Department
Biological Sciences
First Advisor
Joan Lin-Cereghino
First Committee Member
Craig Vierra
Second Committee Member
Douglas C. Weiser
Third Committee Member
Geoff Lin-Cereghino
Abstract
The methylotropic yeast Pichia pastoris has been used for many applications, particularly for its ability to produce and readily secrete heterologous proteins. Nonetheless, there are obstacles in making this useful yeast into a more efficient secretion system that readily secretes problem proteins. In the Lin-Cereghino lab, mutant strains were developed by the method of restriction enzyme mediated integration. These mutants have the ability to secrete β-galactosidase at higher levels in comparison to the wild type. This study focused on characterizing the specific mutant ah2 for its ability to secrete HRP, SLPI, and CALB lipase proteins, as well as using transmission electron microscopy to observe the effect of the pREMI-Z mutation on the morphology. Analysis of the Ah2 protein resulted in a comparative β-galactosidase secretion study, as well as a growth rate study, between the original pREMI-Z ah2 mutant and ah2 mutant cells that were transformed with pKanB-AH2 rescue construct. Lastly, a cell localization experiment was done to examine where Ah2p localizes. By these analyses, we gain a bit more understanding of the P. pastoris secretion pathway, while also outlining a procedure by which to characterize the other pREMI-Z mutants.
Pages
96
Recommended Citation
Bulahan, Rhobe Justine Artates. (2012). Characterizing potential secretion components that increase secretion of recombinant proteins in Pichia Pastoris. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/812
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