The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-1843 |
| Date | 01 January 2013 |
| Creators | Campos, Katherine Helen de Sa |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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