Abstract
G-protein-coupled receptors (GPCRs) are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of GPCRs’ structure and function remains limited. The first bottleneck in GPCR studies is producing sufficient quantities of soluble, functional, and stable receptors. Currently, GPCR production largely depends on the choice of the overexpression host system and the type of detergent used to extract the GPCR from the cell membrane and stabilize the protein outside the membrane bilayer. Here, we present three protocols that we employ in our lab to produce and solubilize stable GPCRs by cell-free in vitro translation systems, HEK cells, and Escherichia coli. Stable receptors can be purified using immunoaffinity chromatography and gel filtration and can be analyzed with standard biophysical techniques and biochemical assays.
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
Klabunde T, Hessler G (2002) Drug design strategies for targeting G-protein-coupled receptors. ChemBioChem 3:928–944
Lundstrom K (2005) Structural biology of G protein-coupled receptors. Bioorg Med Chem Lett 15:3654–3657
Palczewski K, Kumasaka T, Hori T et al (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745
Klammt C, Schwarz D, Eifler N et al (2007) Cell-free production of G protein-coupled receptors for functional and structural studies. J Struct Biol 158:482–493
Reeves PJ, Thurmond RL, Khorana HG (1996) Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines. Proc Natl Acad Sci U S A 93:11487–11492
Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298
Wagner S, Klepsch MM, Schlegel S et al (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105:14371–14376
Hampe W, Voss RH, Haase W et al (2000) Engineering of a proteolytically stable human beta(2)-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus infected insect cells. J Biotechnol 77:219–234
Mouillac B, Caron M, Bonin H et al (1992) Agonist-modulated palmitoylation of b2-adrenergic receptor in Sf9 cells. J Biol Chem 267:21733–21737
Panneels V, Sinning I (2010) Membrane protein expression in the eyes of transgenic flies. Methods Mol Biol 601:135–147
Zhang L, Salom D, He JH et al (2005) Expression of functional G protein-coupled receptors in photoreceptors of transgenic Xenopus laevis. Biochemistry 44:14509–14518
Li N, Salom D, Zhang L et al (2007) Heterologous expression of the adenosine A1 receptor in transgenic mouse retina. Biochemistry 46:8350–8359
Sarramegna V, Muller I, Mousseau G et al (2005) Solubilization, purification and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris. Protein Expr Purif 43:85–93
Dowell SJ, Brown AJ (2002) Yeast assays for G-protein coupled receptors. Receptors Channels 8:343–352
Grisshammer R, Duckworth R, Henderson R (1993) Expression of a rat neurotensin receptor in Escherichia coli. Biochem J 295:571–576
Sarramegna V, Talmont F, Demange P et al (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 60:1529–1546
McCusker EC, Bane SE, O’Malley MA et al (2007) Heterologous GPCR expression: a bottleneck to obtaining crystal structures. Biotechnol Prog 23:540–547
Tate CG, Grisshammer R (1996) Heterologous expression of G-protein-coupled receptors. Trends Biotechnol 14:426–430
Ishihara G, Goto M, Saeki M et al (2005) Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors. Protein Expr Purif 41:27–37
Corin K, Baaske P, Ravel DB et al (2011) A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors. PLoS ONE 6:e23036
Corin K, Baaske P, Ravel DB et al (2011) Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems. PLoS ONE 6:e25067
Wang XQ, Corin K, Baaske P et al (2011) Peptide surfactants for cell-free production of functional G protein-coupled receptors. Proc Natl Acad Sci U S A 108:9049–9054
Koutsopoulos S, Kaiser L, Eriksson HM et al (2012) Designer peptide surfactants stabilize diverse functional membrane proteins. Chem Soc Rev 41:1721–1728
Ren H, Yu D, Ge B et al (2009) High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1. PLoS ONE 4:e4509
Gubellini F, Verdon G, Karpowich NK et al (2011) Physiological response to membrane protein overexpression in E. coli. Mol Cell Proteomics 10:1–17
King K, Dohlman HG, Thorner J et al (1990) Control of yeast mating signal transduction by a mammalian beta-2-adrenergic receptor and Gs alpha-subunit. Science 250:121–123
Corin K, Baaske P, Geissler S et al (2011) Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1. Sci Rep 1:172
Corin K, Pick H, Baaske P et al (2012) Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs. Mol Biosyst 8:1750–1759
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Corin, K., Tegler, L.T., Koutsopoulos, S. (2014). G-Protein-Coupled Receptor Expression and Purification. In: Labrou, N. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 1129. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-977-2_33
Download citation
DOI: https://doi.org/10.1007/978-1-62703-977-2_33
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-976-5
Online ISBN: 978-1-62703-977-2
eBook Packages: Springer Protocols