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Date of Award

2005

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Joan Lin-Cereghino

First Committee Member

Geoff Lin-Cereghino

Second Committee Member

Gregg D. Jongeward

Third Committee Member

David Thomas

Fourth Committee Member

Craig Vierra

Fifth Committee Member

Paul A. Richmond

Abstract

Pichia pastoris is a methylotrophic yeast gaining acclamation for its capabili ties in heterologous protein expression. In contrast to other host organisms such as bacteria or mammalian cells, P. pastoris offers many advantages over its counterparts. For example, P. pastoris is cost-effective in that it can grow to high cell densities on simple media. The optional use of a constitutive (GAP) or inducible (A OXI) promoter and the ab ility to perfo1m post-translational protein modifications are additional qualities that make for a powerful heterologous expression system. This study focuses on harnessing the benefits described to develop a high-throughput reporter system for the screening of potential super-secreting mutant strains of P. pastoris. Plasmid constructs were engineered with the lacZ reporter gene, which encodes for the β-galactosidase protein, and fused to the S. cerevisiae MATa signal sequence. Expression plasmids were successfully transformed in P. pastoris strain yGS 115 followed by induction. Western blot analyses confirm the expression of β-galactosidase and colorimetric activity assays further validate enzymatic function. A mutant library containing cis- and/or trans-acting mutations was created by treating P. pas loris clones harboring the β-galactosidase expression plasmid with ultraviolet (UV) radiation. A colorimetric plate assay was combined with a replica-plating system that enabled the screening of thousands of potential super-secreting mutant colonies in a high-throughput format. This study sheds light onto our current understanding of secretion in yeast and further contributes to developing P. pastoris into a valuable heterologous protein expression system.

Pages

86

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