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Date of Award
2015
Document Type
Thesis - Pacific Access Restricted
Degree Name
Master of Science (M.S.)
Department
Biological Sciences
First Advisor
Geoff Lin-Cereghino
First Committee Member
Douglas Risser
Second Committee Member
Jerry Tsai
Abstract
The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation with rapid growth to high cell densities and provides complex posttranslational modifications. The most successful and commonly used secretion signal leader in Pichia pastoris has been the MAT α prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently even with the MAT α prepro secretion signal. Some strategies to enhance secretion efficiency involved modifying the secretion signal leader. Based on the knob-socket model and Jpred3 ( a secondary structure predictor), eleven deletions of MAT α prepro secretion signal and one MAT α pre double pro-peptide mutant was engineered and assayed with either horseradish peroxidase (HRP), or Candida antarctica lipase B reporter protein to evaluate the correlation between secondary structure and secretion level. In addition, structural analysis through circular dichroism was completed for the wild type pro-peptide and a mutant pro-peptide to evaluate differences in secondary structure. Results suggest pro-peptide amino acids 75-78 play an important role in determining secretion level and a higher secretion level tends to associate with secondary structures that are less defined. With these analyses, optimization of secretion systems can be achieved to impact the fields of science, industry, healthcare, and economics worldwide.
Pages
107
ISBN
9781321660166
Recommended Citation
Wei, Peter. (2015). The structural characterization of the Saccharomyces cerevisiae alpha mating factor secretion signal for recombinant protein secretion in Pichia pastoris. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/177
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