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Date of Award


Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)


Biological Sciences

First Advisor

Joan Lin-Cereghino

First Committee Member

Craig Vierra

Second Committee Member

Douglas C. Weiser

Third Committee Member

Geoff Lin-Cereghino


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.



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