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Date of Award
2016
Document Type
Thesis - Pacific Access Restricted
Degree Name
Master of Science (M.S.)
Department
Biological Sciences
First Advisor
Joan Lin-Cereghino
First Committee Member
Douglas Risser
Second Committee Member
Jerry Tsai
Abstract
The methylotrophic yeast, Pichia pastoris, is the most successful and favored microbial eukaryotic expression system for the production of recombinant proteins for biopharmaceutical or industrial purposes. P. pastoris has the ability to produce foreign proteins at high levels extracellularly, and since it secretes few endogenous proteins, this ability eliminates the need for expensive purification costs. It also combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most commonly utilized secretion signal leader in P. pastoris is the MATα prepro signal leader, originally found in S. cerevisiae. However, because some proteins cannot be secreted efficiently by P. pastoris, strategies to enhance secretion efficiency have involved the modification of the MATα prepro secretion signal leader. The study focuses on using site-directed mutagenesis of specific sets of amino acids of MATα prepro secretion leader to evaluate the correlation between secondary structure and secretion level. MATα pro-HRP mutants were created, in order to analyze the export of heterologous proteins in P. pastoris. In addition, structural analysis through circular dichroism was performed on mutant MATα pro-peptides to evaluate differences in secondary structure as a result of the mutagenesis. Mutants, pSC6 (Δ57-65) and pSC7 (Δ66-70) did not generate the same HRP secretion level as Δ57-70. In addition, a new proposed model of MATα pro-peptide signal leader was created. This new model suggests that the N and C terminus of MATα pro-peptide need to be presented correctly for proper interaction with secretion machinery and for efficient protein secretion. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.
Pages
98
ISBN
9781339960340
Recommended Citation
Chahal, Sabreen. (2016). Structural characterization of the MATα prepro-peptide secretion leader in Pichia pastoris. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/166
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