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Membrane Proteins Production for Structural Analysis pp 71–86Cite as

Bacterial Expression and Stabilization of GPCRs

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Abstract

Although expression of G protein-coupled receptors in heterologous systems such as yeast, insect, and mammal cells has proven to be highly useful for structural studies, bacterial systems have still an interest, in particular for specific purposes such as isotopic labeling in nuclear magnetic resonance analyses. Different approaches have been developed during the past years that consist in either expressing the receptor in bacterial membranes or in functional refolding from inclusion bodies. Here, we review the most recent advances in functional production and folding of GPCRs overexpressed in Escherichia coli and the subsequent stabilization of their native fold in solution.

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References

  • Andrell J, Tate CG (2013) Overexpression of membrane proteins in mammalian cells for structural studies. Mol Membr Biol 30(1):52–63

    Article  PubMed Central  PubMed  Google Scholar 

  • Arcemisbehere L, Sen T, Boudier L, Balestre MN, Gaibelet G, Detouillon E, Orcel H, Mendre C, Rahmeh R, Granier S, Vives C, Fieschi F, Damian M, Durroux T, Baneres JL, Mouillac B (2010) Leukotriene BLT2 receptor monomers activate the G(i2) GTP-binding protein more efficiently than dimers. J Biol Chem 285(9):6337–6347

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Baneres 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(4):801–814

    Article  CAS  PubMed  Google Scholar 

  • Baneres 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(21):20253–20260

    Article  CAS  PubMed  Google Scholar 

  • Bayburt TH, Sligar SG (2010) Membrane protein assembly into Nanodiscs. FEBS Lett 584(9):1721–1727

    Article  CAS  PubMed  Google Scholar 

  • Bayburt TH, Leitz AJ, Xie G, Oprian DD, Sligar SG (2007) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J Biol Chem 282(20):14875–14881

    Article  CAS  PubMed  Google Scholar 

  • Bazzacco P, Billon-Denis E, Sharma KS, Catoire LJ, Mary S, Le Bon C, Point E, Baneres JL, Durand G, Zito F, Pucci B, Popot JL (2012) Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance. Biochemistry 51(7):1416–1430

    Article  CAS  PubMed  Google Scholar 

  • Bosse M, Thomas L, Hassert R, Beck-Sickinger AG, Huster D, Schmidt P (2011) Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding. Biochemistry 50(45):9817–9825

    Article  CAS  PubMed  Google Scholar 

  • Catoire LJ, Damian M, Giusti F, Martin A, van Heijenoort C, Popot JL, Guittet E, Baneres JL (2010) Structure of a GPCR ligand in its receptor-bound state: leukotriene B4 adopts a highly constrained conformation when associated to human BLT2. J Am Chem Soc 132(26):9049–9057

    Article  CAS  PubMed  Google Scholar 

  • Chae PS, Rasmussen SG, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S, Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B, Kobilka B, Gellman SH (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7(12):1003–1008

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chowdhury A, Feng R, Tong Q, Zhang Y, Xie XQ (2012) Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli. Protein Expr Purif 83(2):128–134

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cooke RM, Koglin M, Errey JC, Marshall FH (2013) Preparation of purified GPCRs for structural studies. Biochem Soc Trans 41(1):185–190

    Article  CAS  PubMed  Google Scholar 

  • Corin K, Baaske P, Ravel DB, Song J, Brown E, Wang X, Wienken CJ, Jerabek-Willemsen M, Duhr S, Luo Y, Braun D, Zhang S (2011) Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems. PLoS One 6(11):e25067

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dahmane T, Damian M, Mary S, Popot JL, Baneres JL (2009) Amphipol-assisted in vitro folding of G protein-coupled receptors. Biochemistry 48(27):6516–6521

    Article  CAS  PubMed  Google Scholar 

  • Dahmane T, Rappaport F, Popot JL (2013) Amphipol-assisted folding of bacteriorhodopsin in the presence or absence of lipids: functional consequences. Eur Biophys J 42(2–3):85–101

    Article  CAS  PubMed  Google Scholar 

  • Damian M, Perino S, Polidori A, Martin A, Serre L, Pucci B, Baneres JL (2007) New tensio-active molecules stabilize a human G protein-coupled receptor in solution. FEBS Lett 581(10):1944–1950

    Article  CAS  PubMed  Google Scholar 

  • Damian M, Marie J, Leyris JP, Fehrentz JA, Verdie P, Martinez J, Baneres JL, Mary S (2012) High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs. J Biol Chem 287(6):3630–3641

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • El Moustaine D, Granier S, Doumazane E, Scholler P, Rahmeh R, Bron P, Mouillac B, Baneres JL, Rondard P, Pin JP (2012) Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G-protein coupling. Proc Natl Acad Sci U S A 109(40):16342–16347

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hofmann KP, Scheerer P, Hildebrand PW, Choe HW, Park JH, Heck M, Ernst OP (2009) A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem Sci 34(11):540–552

    Article  CAS  PubMed  Google Scholar 

  • Huang KS, Bayley H, Liao MJ, London E, Khorana HG (1981) Refolding of an integral membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin and two proteolytic fragments. J Biol Chem 256(8):3802–3809

    CAS  PubMed  Google Scholar 

  • Inagaki S, Ghirlando R, White JF, Gvozdenovic-Jeremic J, Northup JK, Grisshammer R (2012) Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid. J Mol Biol 417(1–2):95–111

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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(50):16077–16084

    Article  CAS  PubMed  Google Scholar 

  • Kiefer H, Vogel R, Maier K (2000) Bacterial expression of G-protein-coupled receptors: prediction of expression levels from sequence. Receptors Channels 7(2):109–119

    CAS  PubMed  Google Scholar 

  • Kuszak AJ, Pitchiaya S, Anand JP, Mosberg HI, Walter NG, Sunahara RK (2009) Purification and functional reconstitution of monomeric mu-opioid receptors: allosteric modulation of agonist binding by Gi2. J Biol Chem 284(39):26732–26741

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7(4):339–357

    Article  PubMed  Google Scholar 

  • Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AG, Tate CG (2011) Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474(7352):521–525

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Marullo S, Delavier-Klutchko C, Eshdat Y, Strosberg AD, Emorine L (1988) Human beta 2-adrenergic receptors expressed in Escherichia coli membranes retain their pharmacological properties. Proc Natl Acad Sci U S A 85(20):7551–7555

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mary S, Damian M, Louet M, Floquet N, Fehrentz JA, Marie J, Martinez J, Baneres JL (2012) Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor. Proc Natl Acad Sci U S A 109(21):8304–8309

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mary S, Fehrentz JA, Damian M, Gaibelet G, Orcel H, Verdie P, Mouillac B, Martinez J, Marie J, Baneres JL (2013) Heterodimerization with Its splice variant blocks the ghrelin receptor 1a in a non-signaling conformation: a study with a purified heterodimer assembled into lipid discs. J Biol Chem 288(34):24656–24665

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • McKibbin C, Farmer NA, Jeans C, Reeves PJ, Khorana HG, Wallace BA, Edwards PC, Villa C, Booth PJ (2007) Opsin stability and folding: modulation by phospholipid bicelles. J Mol Biol 374(5):1319–1332

    Article  CAS  PubMed  Google Scholar 

  • Michalke K, Graviere ME, Huyghe C, Vincentelli R, Wagner R, Pattus F, Schroeder K, Oschmann J, Rudolph R, Cambillau C, Desmyter A (2009) Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies. Anal Biochem 386(2):147–155

    Article  CAS  PubMed  Google Scholar 

  • Michalke K, Huyghe C, Lichiere J, Graviere ME, Siponen M, Sciara G, Lepaul I, Wagner R, Magg C, Rudolph R, Cambillau C, Desmyter A (2010) Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1. Anal Biochem 401(1):74–80

    Article  CAS  PubMed  Google Scholar 

  • Miller D, Charalambous K, Rotem D, Schuldiner S, Curnow P, Booth PJ (2009) In vitro unfolding and refolding of the small multidrug transporter EmrE. J Mol Biol 393(4):815–832

    Article  CAS  PubMed  Google Scholar 

  • 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(3):289–298

    Article  CAS  PubMed  Google Scholar 

  • Mitra N, Liu Y, Liu J, Serebryany E, Mooney V, DeVree BT, Sunahara RK, Yan EC (2013) Calcium-dependent ligand binding and G-protein signaling of family B GPCR parathyroid hormone 1 receptor purified in nanodiscs. ACS Chem Biol 8(3):617–625

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Muller I, Sarramegna V, Renault M, Lafaquiere V, Sebai S, Milon A, Talmont F (2008) The full-length mu-opioid receptor: a conformational study by circular dichroism in trifluoroethanol and membrane-mimetic environments. J Membr Biol 223(1):49–57

    Article  CAS  PubMed  Google Scholar 

  • Park SH, Casagrande F, Chu M, Maier K, Kiefer H, Opella SJ (2012a) Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination. Biochim Biophys Acta 1818(3):584–591

    Article  CAS  Google Scholar 

  • Park SH, Das BB, Casagrande F, Tian Y, Nothnagel HJ, Chu M, Kiefer H, Maier K, De Angelis AA, Marassi FM, Opella SJ (2012b) Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491(7426):779–783

    CAS  Google Scholar 

  • Pocanschi CL, Dahmane T, Gohon Y, Rappaport F, Apell HJ, Kleinschmidt JH, Popot JL (2006) Amphipathic polymers: tools to fold integral membrane proteins to their active form. Biochemistry 45(47):13954–13961

    Article  CAS  PubMed  Google Scholar 

  • Pocanschi CL, Popot JL, Kleinschmidt JH (2013) Folding and stability of outer membrane protein A (OmpA) from Escherichia coli in an amphipathic polymer, amphipol A8-35. Eur Biophys J 42(2–3):103–118

    Article  CAS  PubMed  Google Scholar 

  • Popot JL (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem 79:737–775

    Article  CAS  PubMed  Google Scholar 

  • Popot JL, Gerchman SE, Engelman DM (1987) Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process. J Mol Biol 198(4):655–676

    Article  CAS  PubMed  Google Scholar 

  • Proverbio D, Roos C, Beyermann M, Orban E, Dotsch V, Bernhard F (2013) Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments. Biochim Biophys Acta 1828(9):2182–2192

    Article  CAS  PubMed  Google Scholar 

  • Rahmeh R, Damian M, Cottet M, Orcel H, Mendre C, Durroux T, Sharma KS, Durand G, Pucci B, Trinquet E, Zwier JM, Deupi X, Bron P, Baneres JL, Mouillac B, Granier S (2012) Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy. Proc Natl Acad Sci U S A 109(17):6733–6738

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ren H, Yu D, Ge B, Cook B, Xu Z, Zhang S (2009) High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1. PLoS One 4(2):e4509

    Article  PubMed Central  PubMed  Google Scholar 

  • Schlinkmann KM, Hillenbrand M, Rittner A, Kunz M, Strohner R, Pluckthun A (2012a) Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution. J Mol Biol 422(3):414–428

    Article  CAS  Google Scholar 

  • Schlinkmann KM, Honegger A, Tureci E, Robison KE, Lipovsek D, Pluckthun A (2012b) Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations. Proc Natl Acad Sci U S A 109(25):9810–9815

    Article  CAS  Google Scholar 

  • Schlinkmann KM, Pluckthun A (2013) Directed evolution of G-protein-coupled receptors for high functional expression and detergent stability. Methods Enzymol 520:67–97

    Article  CAS  PubMed  Google Scholar 

  • Schmidt P, Lindner D, Montag C, Berndt S, Beck-Sickinger AG, Rudolph R, Huster D (2009) Prokaryotic expression, in vitro folding, and molecular pharmacological characterization of the neuropeptide Y receptor type 2. Biotechnol Prog 25(6):1732–1739

    CAS  PubMed  Google Scholar 

  • Schroder-Tittmann K, Bosse-Doenecke E, Reedtz-Runge S, Ihling C, Sinz A, Tittmann K, Rudolph R (2010) Recombinant expression, in vitro refolding, and biophysical characterization of the human glucagon-like peptide-1 receptor. Biochemistry 49(36):7956–7965

    Article  PubMed  Google Scholar 

  • Scott DJ, Kummer L, Tremmel D, Pluckthun A (2013) Stabilizing membrane proteins through protein engineering. Curr Opin Chem Biol 17(3):427–435

    Article  CAS  PubMed  Google Scholar 

  • Shiroishi M, Kobayashi T, Ogasawara S, Tsujimoto H, Ikeda-Suno C, Iwata S, Shimamura T (2011) Production of the stable human histamine H(1) receptor in Pichia pastoris for structural determination. Methods 55(4):281–286

    Article  CAS  PubMed  Google Scholar 

  • Skretas G, Georgiou G (2009) Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor. Biotechnol Bioeng 102(2):357–367

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Skretas G, Makino T, Varadarajan N, Pogson M, Georgiou G (2012) Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli. Metab Eng 14(5):591–602

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tate CG, Schertler GF (2009) Engineering G protein-coupled receptors to facilitate their structure determination. Curr Opin Struct Biol 19(4):386–395

    Article  CAS  PubMed  Google Scholar 

  • Thompson AA, Liu JJ, Chun E, Wacker D, Wu H, Cherezov V, Stevens RC (2011) GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles. Methods 55(4):310–317

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci U S A 93(26):15047–15050

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tucker J, Grisshammer R (1996) Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem J 317(Pt 3):891–899

    CAS  PubMed Central  PubMed  Google Scholar 

  • Verardi R, Traaseth NJ, Masterson LR, Vostrikov VV, Veglia G (2012) Isotope labeling for solution and solid-state NMR spectroscopy of membrane proteins. Adv Exp Med Biol 992:35–62

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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(7203):486–491

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Weiss HM, Grisshammer R (2002) Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli. Eur J Biochem/FEBS 269(1):82–92

    Article  CAS  Google Scholar 

  • White JF, Noinaj N, Shibata Y, Love J, Kloss B, Xu F, Gvozdenovic-Jeremic J, Shah P, Shiloach J, Tate CG, Grisshammer R (2012) Structure of the agonist-bound neurotensin receptor. Nature 490(7421):508–513

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Whorton MR, Bokoch MP, Rasmussen SG, Huang B, Zare RN, Kobilka B, Sunahara RK (2007) A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc Natl Acad Sci U S A 104(18):7682–7687

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wiktor M, Morin S, Sass HJ, Kebbel F, Grzesiek S (2013) Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor. J Biomol NMR 55(1):79–95

    Article  CAS  PubMed  Google Scholar 

  • Witte K, Kaiser A, Schmidt P, Splith V, Thomas L, Berndt S, Huster D, Beck-Sickinger AG (2013) Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration. Biol Chem 394(8):1045–1056

    Article  CAS  PubMed  Google Scholar 

  • Yeliseev AA, Wong KK, Soubias O, Gawrisch K (2005) Expression of human peripheral cannabinoid receptor for structural studies. Protein Sci 14(10):2638–2653

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zaccai NR, Yunus K, Matthews SM, Fisher AC, Falconer RJ (2007) Refolding of a membrane protein in a microfluidics reactor. Eur Biophys J 36(6):581–588

    Article  CAS  PubMed  Google Scholar 

  • Zhao X, Nagai Y, Reeves PJ, Kiley P, Khorana HG, Zhang S (2006) Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin. Proc Natl Acad Sci U S A 103(47):17707–17712

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zoonens M, Giusti F, Zito F, Popot JL (2007) Dynamics of membrane protein/amphipol association studied by Forster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins. Biochemistry 46(36):10392–10404

    Article  CAS  PubMed  Google Scholar 

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  1. Institute of Biomolecules Max Mousseron, UMR-CNRS 5247, Faculty of Pharmacy, Université Montpellier 1 and 2, 15 avenue Charles Flahault, BP 14491, 34093, Montpellier, France

    Jean-Louis Banères

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  1. Jean-Louis Banères
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Correspondence to Jean-Louis Banères .

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  1. UMR-CNRS 7275, Institute of Molecular and Cellular Pharmacology, Valbonne, France

    Isabelle Mus-Veteau

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Banères, JL. (2014). Bacterial Expression and Stabilization of GPCRs. In: Mus-Veteau, I. (eds) Membrane Proteins Production for Structural Analysis. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-0662-8_3

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