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Date of Award
2014
Document Type
Thesis - Pacific Access Restricted
Degree Name
Master of Science (M.S.)
Department
Biological Sciences
First Advisor
Joan Lin-Cereghino
First Committee Member
Kristin Kohler
Second Committee Member
Der Thor
Abstract
In effort to understand and isolate genes that are associated with protein secretion, the Lin-Cereghino laboratory at University of the Pacific created mutant strains of Pichia pastoris using the restriction enzyme mediated integration method. The mutants exhibited an unusual ability to supersecrete beta-galactosidase, due to the effects of a randomly disrupted gene by pREMI-Z. To learn more about the novel effects of the gene disruption, nine beta-galactosidase supersecreters ( bgs ) have been characterized for their phenotypes such as growth rate, cell wall integrity, and ability to produce and secrete various types of recombinant proteins. The mutants showed various population doubling times, which ranged from 1.7 to 2.4 hours. Generally, the mutants with severely diminished growth rates had much lower secretion of the reporter proteins. The mutants also showed different levels of cell wall (osmotic) defect, indicated by moderate to severe leakage of alkaline phosphatase from the vacuole. It was revealed that the cell wall defect was not necessarily associated with increased protein secretion, which suggests that the cell wall may not be a limiting barrier for the secretion of most reporter proteins. The result of the reporter study suggests that the secretion phenotypes of bgs mutants were protein specific and likely to be dependent upon the structure of the secreted protein rather than the size.
Pages
58
ISBN
9781321359961
Recommended Citation
Weaver, Jun Eon. (2014). Characterizing phenotypes of Pichia pastoris mutants that show enhanced secretion of recombinant proteins. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/188
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