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Date of Award
2009
Document Type
Thesis - Pacific Access Restricted
Degree Name
Master of Science (M.S.)
Department
Biological Sciences
First Advisor
Geoff Lin-Cereghino
First Committee Member
Joan Lin-Cereghino
Second Committee Member
Srinivas Venkatram
Third Committee Member
Craig A. Vierra
Abstract
The unicellular yeast, Pichia pastoris has currently emerged as one of the most popular host systems for heterologous proteins due to its relatively cheap cost, easy genetic manipulability, ability to perform post-translational modifications on proteins, and respiratory growth capabilities which allow it to be cultured in very high concentrations. Over 700 foreign proteins have been recombinantly expressed using P. pastoris.
Although P. pastoris appears to be an ideal host system, its main drawback is its inability to efficiently export some heterologous proteins into the extracellular medium. The incorporation of S. cerevisiae's MATα pre-pro signal leader (MATα) has led to increased protein secretion in most cases. MATα is thus used in the production of 90% of all proteins secreted in P. pastoris. However secretion efficiency still remains a problem.
It has been suspected that low secretion may be attributed to improper extracellular targeting (a function of MATα). In order to address these issues there has been a precedent for performing limited mutagenesis of a signal leader peptide (like MATα) to increase protein secretion. In one study the insertion of a 10 amino-acid residue into MATα resulted in a 5-fold increase in secretion of bacterial phytase, an important industrial enzyme. Despite this success there have been no systematic mutagenesis processes which would help elucidate the reason behind this case of increased secretion.
In our study, we performed a series of mutagenesis events, both random and site directed, with the intent of illuminating the mechanisms of MATα that contribute to secretion. As a result were able to create a novel secretion signal (pLL3) with enhanced secretion levels of our reporter protein HRP.
Pages
86
Recommended Citation
Kim, Daniel. (2009). Characterization of the MATα pre-/pro- peptide by mutagenesis as a means to optimize secretion in pichia pistoris. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/738
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