Abstract
A key point when it comes to heterologous expression of eukaryotic membrane proteins (EMPs) is the choice of the best-suited expression platform. The yeast Pichia pastoris has proven to be a very versatile system showing promising results in a growing number of cases. Indeed, its particular methylotrophic characteristics combined to the very simple handling of a eukaryotic microorganism that possesses the majority of mammalian-like machineries make it a very competitive expression system for various complex proteins, in amounts compatible with functional and structural studies. This chapter describes a set of robust methodologies routinely used for the successful expression of a variety of EMPs, going from yeast transformation with the recombinant plasmid to the analysis of the quality and quantity of the proteins produced.
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
References
Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54(6):741–750
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Sarramegna V, Talmont F, Demange P, Milon A (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification. Cell Mol Life Sci 60(8):1529–1546
Alkhalfioui F, Logez C, Bornert O, Wagner R (2011) Expression systems: Pichia pastoris. In: Robinson AS (ed) Production of membrane proteins–strategies for expression and isolation. Wiley-VCH, Weinheim. doi:10.1002/9783527634521
Bill RM (2014) Playing catch-up with Escherichia coli: using yeast to increase success rates in recombinant protein production experiments. Front Microbiol 5:85
Bertheleme N, Singh S, Dowell S, Byrne B (2015) Heterologous expression of G-protein-coupled receptors in yeast. Methods Enzymol 556:141–164
André N, Cherouati N, Prual C, Steffan T, Zeder-Lutz G, Magnin T, Pattus F, Michel H, Wagner R, Reinhart C (2006) Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Sci 15:1115–1126
Magnin T, Fiez-Vandal C, Potier N, Coquard A, Leray I, Steffan T, Logez C, Alkhalfioui F, Pattus F, Wagner R (2009) A novel, generic and effective method for the rapid purification of G protein-coupled receptors. Protein Expr Purif 64(1):1–7
Bornert O, Møller TC, Boeuf J, Candusso MP, Wagner R, Martinez KL, Simonin F (2013) Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors. PLoS One 8(2):e56336
Logez C, Berger S, Legros C, Banères JL, Cohen W, Delagrange P, Nosjean O, Boutin JA, Ferry G, Simonin F, Wagner R (2014) Recombinant human melatonin receptor MT1 isolated in mixed detergents shows pharmacology similar to that in mammalian cell membranes. PLoS One 9(6):e100616
Logez C, Alkhalfioui F, Byrne B, Wagner R (2012) Preparation of Pichia pastoris expression plasmids. Methods Mol Biol 866:25–40
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. Methods Mol Biol 866:197–207
Bernier V, Bichet DG, Bouvier M (2004) Pharmacological chaperone action on G-protein-coupled receptors. Curr Opin Pharmacol 4(5):528–533
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Hartmann, L., Kugler, V., Wagner, R. (2016). Expression of Eukaryotic Membrane Proteins in Pichia pastoris . In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 1432. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3637-3_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3637-3_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3635-9
Online ISBN: 978-1-4939-3637-3
eBook Packages: Springer Protocols