Abstract
The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.
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References
Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52
Porro D, Gasser B, Fossati T, Maurer M, Branduardi P, Sauer M, Mattanovich D (2011) Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems? Appl Microbiol Biotechnol 89:939–48
Sevastsyanovich Y, Alfasi S, Cole J (2009) Recombinant protein production: a comparative view on host physiology. N Biotechnol 25:175–80
Cole J, Ferrer P, Mattanovich D, Archer D (2013) Recombinant protein production 6: a comparative view on host physiology. N Biotechnol 30:246
Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. N Biotechnol 30(4):385–404
Näätsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS, Glieder A (2012) Deletion of the Pichia pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic biology. PLoS One 7:e39720
Vogl T, Hartner FS, Glieder A (2013) New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris. Curr Opin Biotechnol 24(6):1094–101
Lee CC, Williams TG, Wong DWS, Robertson GH (2005) An episomal expression vector for screening mutant gene libraries in Pichia pastoris. Plasmid 54:80–5
Lee CC, Wong DWS, Robertson GH (2005) Cloning and characterization of the xyn11A gene from Lentinula edodes. Protein J 24:21–6
Sasagawa T, Matsui M, Kobayashi Y, Otagiri M, Moriya S, Sakamoto Y, Ito Y, Lee CC, Kitamoto K, Arioka M (2011) High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors. Plasmid 65:65–9
Weis R, Luiten R, Skranc W, Schwab H, Wubbolts M, Glieder A (2004) Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res 5:179–89
Liu Z, Pscheidt B, Avi M, Gaisberger R, Hartner FS, Schuster C, Skranc W, Gruber K, Glieder A (2008) Laboratory evolved biocatalysts for stereoselective syntheses of substituted benzaldehyde cyanohydrins. Chembiochem 9:58–61
Weinhandl K, Winkler M, Glieder A, Camattari A (2012) A novel multi-enzymatic high throughput assay for transaminase activity. Tetrahedron 68:7586–7590
Gudiminchi RK, Geier M, Glieder A, Camattari A (2013) Screening for cytochrome P450 expression in Pichia pastoris whole cells by P450-carbon monoxide complex determination. Biotechnol J 8:146–52
Forni L, Mora-Arellano V, Packer J, Wilson R (1986) Nitrogen dioxide and related free radicals: electron-transfer reactions with organic compounds in solutions containing nitrite or nitrate. J Chem Soc Perkin Trans 1:1–6
Acknowledgments
This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ); the Federal Ministry of Traffic, Innovation and Technology (bmvit); the Styrian Business Promotion Agency SFG; and the Standortagentur Tirol and ZIT—Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. The research leading to these results has received funding from the European Union’s Seventh Framework Programme FP7/2007–2013 under grant agreement nr. 266025 (BIONEXGEN).
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Camattari, A., Weinhandl, K., Gudiminchi, R.K. (2014). Methods for Efficient High-Throughput Screening of Protein Expression in Recombinant Pichia pastoris Strains. In: Mapelli, V. (eds) Yeast Metabolic Engineering. Methods in Molecular Biology, vol 1152. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0563-8_6
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DOI: https://doi.org/10.1007/978-1-4939-0563-8_6
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-0563-8
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