Abstract
The first bottleneck in olfactory receptor (OR) structural and functional studies is to produce sufficient quantities of soluble, functional, and stable receptors. Other production systems have been used and summarized in other chapters of this book. We here show that commercial cell-free in vitro translation systems can be used to produce milligrams of soluble and functional olfactory receptors within several hours directly from plasmid DNA with select optimal detergents. These olfactory receptors can be purified using immunoaffinity 1D4 monoclonal antibody rhodopsin-tag and gel filtration, and can be analyzed using gel electrophoresis and with other standard techniques. The olfactory receptors and other scent-related receptors produced by the cell-free method fold properly and are able to bind their odorants.
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References
Grisshammer R, Duckworth R, Henderson R (1993) Expression of a rat neurotensin receptor in Escherichia coli. Biochem J 295:571–576
Hampe W et al (2000) Engineering of a proteolytically stable human β2-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
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 60:1529–1546
McCusker EC, Bane SE, O’Malley MA, Robinson AS (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 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
Klammt C et al (2007) Cell-free production of G protein-coupled receptors for functional and structural studies. J Struct Biol 158:482–493
Kaiser L et al (2008) Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses. Proc Natl Acad Sci 105:15726–15731
Corin K et al (2011) A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors. PLoS ONE 6:e23036
Corin K 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 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
Takayama H et al (2008) High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81. PLoS ONE 3:e2314
Cook BL et al (2008) Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line. PLoS ONE 3:e2920
Cook BL et al (2009) Large-scale production and study of a synthetic GPCR: human olfactory receptor 17-4. Proc Natl Acad Sci U S A 106:11925–11930
Wang X et al (2011) Study of two G-protein coupled receptor variants of human trace amine-associated receptor 5. Sci Rep 1:e102
Corin et al (2011) Biochemical study of a bioengineered functional G-protein coupled receptor human vomeronasal type 1 receptor 1. Sci Rep 1:e172
Corin et al (2012) Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs. Mol Biosyst 8:1750–1759
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 93:11487–11492
Acknowledgements
We gratefully acknowledge the initial supported by a generous grant from ROHM, Kyoto, Japan, later in part by DARPA-HR0011-09-C-0012 and Yang Trust Fund.
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Corin, K., Wang, X., Zhang, S. (2014). Production of Olfactory Receptors Using Commercial E. coli Cell-free Systems. In: Park, T. (eds) Bioelectronic Nose. Springer, Dordrecht. https://doi.org/10.1007/978-94-017-8613-3_7
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DOI: https://doi.org/10.1007/978-94-017-8613-3_7
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