Abstract
One of the major advantages of using Pichia pastoris is that it is readily adapted to large-scale culture in bioreactors. Bioreactors allow precise regulation of cell growth parameters increasing both yields and reproducibility of the culture. P. pastoris cultures grow to very high cell densities which helps minimise culture volume and facilitates downstream processing of the sample. Here, we provide protocols for the large-scale production of the human adenosine A2A receptor (A2AR) and provide some details of how bioreactor cultures can be used for optimisation of expression of the human dopamine D2 receptor (D2DR).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cereghino GPL, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Jahic M, Wallberg F, Bollok M, Garcia P, Enfors S-O (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris biorector cultures. Microb Cell Fact 2:6–17
Charoenrat T, Ketudat-Cairns M, Stendahl-Andersen H, Jahic M, Enfors S-O (2005) Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioprocess Biosyst Eng 27:399–406
Jahic M, Veide A, Charoenrat T, Teeri T, Enfors S-O (2006) Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnol Prog 22:1465–1473
Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135
Singh S, Gras A, Fiez-Vandal C, Ruprecht J, Rana R, Martinez M, Strange PG, Wagner R, Byrne B (2008) Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures. Microb Cell Fact 7:28–38
Zhang W, Smith LA, Plantz BA, Schlegel VL, Meagher MM (2002) Design of methanol feed control in Pichia pastoris fermentations based upon a growth model. Biotechnol Prog 18:1392–1399
Northcoate DH, Warne RW (1952) The chemical composition and structure of the yeast cell wall. Biophys J 51:232–236.2
Salazar O, Asenjo JA (2007) Enzymatic lysis of microbial cells. Biotechnol Lett 29:985–994
Acknowledgments
This research was funded by the MepNet consortium, the Biotechnology and Biological Research Council and GlaxoSmithKline.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+business Media, LLC
About this protocol
Cite this protocol
Singh, S., Gras, A., Fiez-Vandal, C., Martinez, M., Wagner, R., Byrne, B. (2012). Large-Scale Production of Membrane Proteins in Pichia pastoris: The Production of G Protein-Coupled Receptors as a Case Study. In: Bill, R. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 866. Humana Press. https://doi.org/10.1007/978-1-61779-770-5_17
Download citation
DOI: https://doi.org/10.1007/978-1-61779-770-5_17
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-769-9
Online ISBN: 978-1-61779-770-5
eBook Packages: Springer Protocols