The methylotrophic yeast, Pichia pastoris, is a popular protein expression system because of its ability to produce high levels of heterologous gene products either intracellularly or extracellularly. Targetting proteins to the extracellullar medium serves as a major purification step because the yeast secretes only a small amount of native protein. Hundreds of recombinant proteins, ranging from spider silk proteins to human insulin, have been produced in this yeast. Despite success of the P. pastoris system, relatively little is known about the molecular biology of its secretion machinery. In order to better understand the mechanisms by which P. pastoris produces and localizes proteins, we have undertaken an analysis of various elements which affect secretion of proteins. We would like to continue projects currently ongoing in our lab: 1. to characterize the genes from isolated mutant strains which secrete higher levels of beta-galactosidase; 2. to understand the molecular nature of leader peptides typically used for secretion of foreign proteins;and 3. to elucidate the nature of the E. coli maltose-binding protein (MBP) as an escort protein and to determine how this bacterial partner protein enhances secretion of fusion proteins in yeast. These studies will identify cis- and trans-acting factors that dictate the export efficiency of proteins expressed in P. pastoris-- illuminating the basic biology behind this secretory system. The elucidation of secretion mechanisms will have two long term benefits: it will increase the basic understanding of how Pichia pastoris targets its proteins, and it will help create a better host system for the growing number of scientists who employ the Pichia pastoris heterologous protein expression as a tool in their own research.

Public Health Relevance

This research will benefit public health by improving a popular yeast protein factory currently used to produce hundreds of proteins useful for both applied and basic research. These include important products such as human collagen, vaccines, and anti-cancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15GM065882-03
Application #
7644756
Study Section
Cell Structure and Function (CSF)
Program Officer
Ainsztein, Alexandra M
Project Start
2002-09-01
Project End
2013-08-31
Budget Start
2009-09-30
Budget End
2013-08-31
Support Year
3
Fiscal Year
2009
Total Cost
$175,391
Indirect Cost
Name
University of the Pacific-Stockton
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
625948831
City
Stockton
State
CA
Country
United States
Zip Code
95211
Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine et al. (2013) Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins. Biotechnol Lett 35:1925-35
Lin-Cereghino, Geoff P; Stark, Carolyn M; Kim, Daniel et al. (2013) The effect of ?-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene 519:311-7
Staley, Chris A; Huang, Amy; Nattestad, Maria et al. (2012) Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris. Gene 496:118-27
Li, Zhiguo; Leung, Wilson; Yon, Amy et al. (2010) Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris. Protein Expr Purif 72:113-24
Li, Zhiguo; Moy, Allison; Gomez, Seth R et al. (2010) An improved method for enhanced production and biological activity of human secretory leukocyte protease inhibitor (SLPI) in Pichia pastoris. Biochem Biophys Res Commun 402:519-24
Li, Zhiguo; Moy, Allison; Sohal, Kirti et al. (2009) Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris. Protein Expr Purif 67:175-81
Hartner, Franz S; Ruth, Claudia; Langenegger, David et al. (2008) Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 36:e76
Lin-Cereghino, Joan; Hashimoto, Matthew D; Moy, Allison et al. (2008) Direct selection of Pichia pastoris expression strains using new G418 resistance vectors. Yeast 25:293-9
Lin-Cereghino, Joan; Wong, William W; Xiong, See et al. (2005) Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques 38:44, 46, 48
Thor, Der; Xiong, See; Orazem, Claire C et al. (2005) Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker. FEMS Yeast Res 5:935-42