Abstract
Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.
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
Junge F, Haberstock S, Roos C, Stefer S, Proverbio D, Dötsch V, Bernhard F (2011) Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins. Nat Biotechnol 28:262–271
Spirin AS (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol 22:538–545
Schneider B, Junge F, Shirokov VA et al (2010) Membrane protein expression in cell-free systems. Methods Mol Biol 601:165–186
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
Yang JP, Cirico T, Katzen F et al (2011) Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs. BMC Biotechnol 11:57
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 X, 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
Kaiser L, Graveland-Bikker J, Steuerwald D et al (2008) Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses. Proc Natl Acad Sci U S A 105:15726–15731
Ritz S, Hulko M, Zerfass C et al (2013) Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs). Biochimie 95:1909–1916
Sansuk K, Balog CI, van der Does AM et al (2008) GPCR proteomics: mass spectrometric and functional analysis of histamine H1 receptor after baculovirus-driven and in vitro cell free expression. J Proteome Res 7:621–629
Junge F, Luh LM, Proverbio D et al (2010) Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor. J Struct Biol 172:94–106
Proverbio D, Roos C, Beyermann M et al (2013) Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments. Biochim Biophys Acta 1828:2182–2192
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
Klammt C, Perrin MH, Maslennikov I et al (2011) Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents. Protein Sci 20:1030–1041
Li Y, Wang E, Wang Y (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein Expr Purif 16:355–358
Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J Am Chem Soc 126:3477–348715
Haberstock S, Roos C, Hoevels Y et al (2012) A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems. Protein Expr Purif 82:308–316
Pédelacq JD, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88
Drew D, Newstead S, Sonoda Y (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3:784–798
Banères JL, Popot JL, Mouillac B (2011) New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322
Breyton C, Gabel F, Abla M et al (2009) Micellar and biochemical properties of (hemi)fluorinated surfactants are controlled by the size of the polar head. Biophys J 97:1077–1086
Lyukmanova EN, Shenkarev ZO, Khabibullina NF et al (2012) Lipid-protein nanodisks for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles, bicelles and liposomes. Biochim Biophys Acta 1818:349–358
Roos C, Zocher M, Müller D et al (2012) Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase. Biochim Biophys Acta 1818:3098–3106
Dahmane T, Damian M, Mary S et al (2009) Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48:6516–6521
Basu D, Castellano JM, Thomas N, Mishra RK (2013) Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform. Biotechnol Prog 29:601–608
Wang X, Cui Y, Wang J (2013) Efficient expression and immunoaffinity purification of human trace amine-associated receptor 5 from E. coli cell-free system. Protein Pept Lett 20:473–480
Gao T, Petrlova J, He W, Huser T et al (2012) Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs. PLoS One 7:e44911
Acknowledgments
This work was supported by the Collaborative Research Centre (SFB) 807 of the German Research Foundation (DFG) and by the Alexander von Humboldt-Foundation. We further thank Vladimir Shirokov for critical discussions.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Orbán, E., Proverbio, D., Haberstock, S., Dötsch, V., Bernhard, F. (2015). Cell-Free Expression of G-Protein-Coupled Receptors. In: Owens, R. (eds) Structural Proteomics. Methods in Molecular Biology, vol 1261. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2230-7_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2230-7_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2229-1
Online ISBN: 978-1-4939-2230-7
eBook Packages: Springer Protocols