Characterization and Analysis of Proteins Secreted by the Mutant Pichia pastoris Strain, bgs13
Date of Award
Master of Science (M.S.)
Geoffrey P. Lin-Cereghino
First Committee Member
Craig A. Vierra
Second Committee Member
Douglas D. Risser
The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for research, clinical, and industrial purposes to produce thousands of recombinant protein products. Because P. pastoris secretes very few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, a disadvantage to the programmed export is that some recombinant proteins intended for secretion are retained within the cell and may be subsequently degraded. A mutant strain isolated in our lab, containing a pREMI-derived disruption of the BGS13 gene, has displayed elevated levels of secretion for a variety of reporter proteins. The wild-type protein expressed from this gene shares homology with the S. cerevisiae Pkc1 protein. It has been elucidated that the Bgs13 protein plays a crucial role in the P. pastoris secretory network, but its specific mode(s) of action is currently unclear.
To reveal the differences in the secretion mechanism and cell wall integrity between wild-type P. pastoris and the bgs13 strain, we identified and characterized differential protein populations found in the extracellular medium and those extracted from the cell wall. The proteomic approach revealed that the bgs13 strain released an increased array of normally secreted proteins, endogenous proteins, and Endoplasmic Reticulum(ER)-resident chaperones. The role of the Bgs13 protein in the Cell Wall Integrity pathway was further investigated by an analysis of the relative cell wall porosity that indicated comparable porosity between the wild-type and bgs13 strains. Because ER-resident chaperones were found to be released in higher abundance by the bgs13 strain, intracellular protein disulfide isomerase levels were analyzed, but no major differences between the two strains were detected. Nonetheless, this result points to a possible occurrence of an unorthodox mode of protein sorting in the bgs13 strain that leads to enhanced release of proteins and cell wall structural changes. This research explicated, in part, the underlying mechanisms of enhanced secretion of the bgs13 strain.
Naranjo, Christopher Alan. (2019). Characterization and Analysis of Proteins Secreted by the Mutant Pichia pastoris Strain, bgs13. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/3610