Abstract
Integral membrane proteins, in particular receptors, regulate numerous physiological functions. The primary difficulty presented by their study in vitro is to obtain them in sufficient amounts in a functional state. Escherichia coli is a host of choice for producing recombinant proteins for structural studies. However, insertion of G-protein coupled receptors into its plasma membrane usually results in bacterial death. An alternative approach consists of targeting recombinant receptors to inclusion bodies, where they accumulate without affecting bacterial growth, and then fold them in vitro . We describe here a general approach that consists of accumulating the receptor in bacterial inclusion bodies, then purifying it under denaturing conditions. A simple assay is then described to screen for refolding conditions of the protein.
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References
Bockaert J, Pin JP (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J 18:1723–1729
Bockaert J, Claeysen S, Becamel C, Pinloche S, Dumuis A (2002) G protein-coupled receptors: dominant players in cell-cell communication. Int Rev Cytol 212:63–132
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745
Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318:1258–1265
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, Kobilka BK (2007) Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450:383–387
Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R, Leslie AG, Tate CG, Schertler GF (2008) Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454:486–491
Kobilka B, Schertler GF (2008) New G-protein-coupled receptor crystal structures: insights and limitations. Trends Pharmacol Sci 29:79–83
Serrano-Vega MJ, Magnani F, Shibata Y, Tate CG (2008) Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci USA 105:877–882
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
Mancia F, Hendrickson WA (2007) Expression of recombinant G-protein coupled receptors for structural biology. Mol Biosyst 3:723–734
Tucker J, Grisshammer R (1996) Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem J 317:891–899
Weiss HM, Grisshammer R (2002) Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli. Eur J Biochem 269:82–92
Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD, Breer H (1996) Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding. Biochemistry 35:16077–16084
Banères JL, Martin A, Hullot P, Girard JP, Rossi JC, Parello J (2003) Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1. J Mol Biol 329:801–814
Banères JL, Mesnier D, Martin A, Joubert L, Dumuis A, Bockaert J (2005) Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy. J Biol Chem 280:20253–20260
Park SH, Prytulla S, De Angelis AA, Brown JM, Kiefer H, Opella SJ (2006) High-resolution NMR spectroscopy of a GPCR in aligned bicelles. J Am Chem Soc 128:7402–7403
Bane SE, Velasquez JE, Robinson AS (2007) Expression and purification of milligram levels of inactive G-protein coupled receptors in E. coli. Protein Expr Purif 52:348–355
Damian M, Martin A, Mesnier D, Pin JP, Baneres JL (2006) Asymmetric conformational changes in a GPCR dimer controlled by G-proteins. EMBO J 25:5693–5702
Kiefer H, Vogel R, Maier K (2000) Bacterial expression of G-protein-coupled receptors: prediction of expression levels from sequence. Receptors Channels 7:109–119
Thai K, Choi J, Franzin CM, Marassi FM (2005) Bcl-XL as a fusion protein for the high-level expression of membrane-associated proteins. Protein Sci 14:948–955
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Mouillac, B., Banères, JL. (2010). Mammalian Membrane Receptors Expression as Inclusion Bodies in Escherichia coli . In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology™, vol 601. Humana Press. https://doi.org/10.1007/978-1-60761-344-2_3
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DOI: https://doi.org/10.1007/978-1-60761-344-2_3
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