Effects of Fetal Bovine Serum on the Proteome and Secretome of Pichia Pastoris

Author

Kenneth L. Nguyen, University of the PacificFollow

Date of Award

2024

Document Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Geoffrey Lin-Cereghino

First Committee Member

Craig Vierra

Second Committee Member

Der Thor

Third Committee Member

Douglas Risser

Abstract

Komagataella pastoris, formerly known as Pichia pastoris, and hereafter referred to as Pichia, is a methylotrophic yeast widely employed as a recombinant protein factory for biotechnical and industrial purposes. P. pastoris boasts the ability to thrive at high cell densities, executes numerous post-translational modifications, and exhibits minimal secretion of endogenous proteins, thus greatly facilitating the expression and purification of recombinant proteins. Despite these advantages, Pichia still presents certain challenges as an expression system. Occasionally, recombinant proteins are retained within the cell and subject to degradation. Furthermore, Pichia falls short in matching the production capabilities of more widely used systems like Escherichia coli in terms of sheer numbers of recombinant proteins generated. When incubating a strain of Pichia expressing reporter protein enhanced green fluorescent protein (eGFP) (yJC100:pDT300) with fetal bovine serum (FBS), western blot analysis revealed a novel, higher molecular band in addition to the expected band, suggesting that FBS was altering recombinant protein expression. The alterations to this higher molecular weight variant were confirmed to happen intracellularly, but the molecular mechanisms behind it remain unclear. To elucidate the intracellular molecular mechanisms behind the production of the novel recombinant protein variant, our lab utilized site-directed mutagenesis and mass spectrometry. Through site-directed mutagenesis, we were able to localize the alteration facilitated by FBS to the C-terminus of eGFP, demonstrating that post-translational modifications incurred by FBS incubation occur at the C-terminus. Analysis of intracellular lysate of yJC100:pDT300 revealed proteomic alteration caused by treating P. pastoris with fetal bovine serum, presenting possible key players in FBS’s interaction with yJC100:pDT300. LC-MS was also used to analyze the extracellular media of FBS-treated yJC100:pDT300, revealing peptide discrepancies in the C-terminus of eGFP between FBS-treated and FBS-untreated samples. These results confirm that FBS’s interaction with yJC100:pDT300 significantly affects secretion through interaction with eGFP’s C-terminus. Our research serves to characterize the FBS’s interaction with yJC100:pDT300 and set precedence for future work in further characterization and optimization of the novel mechanism occurring due to FBS’s presence in growth media.

Pages

93

Recommended Citation

Nguyen, Kenneth L.. (2024). Effects of Fetal Bovine Serum on the Proteome and Secretome of Pichia Pastoris. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/4276