Heterologous Expression of Membrane Proteins for Structural Analysis

  • Isabelle Mus-VeteauEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 601)


Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are involved in numerous diseases, like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer disease. However, studies of these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the heterologous expression systems now available to produce large amounts of MPs for structural proteomics, and describes the strategies that allowed the determination of the structure of the first heterologously expressed mammalian MPs.

Key words

Integral membrane proteins heterologous expression systems solubilization stabilization crystallization 3D structure 



I thank M. Bidet, O. Joubert and R. Nehmé for critical reading of the manuscript and the European Commission through the FP6 specific targeted research project Innovative Tools for Membrane Structural Proteomics (LSH-2003-1.1.0-1) that financially supported several authors of this volume for their research on heterologous expression systems, new surfactant molecules and MP stabilization.


  1. 1.
    Liu J, Rost B (2001) Comparing function and structure between entire proteomes. Protein Sci 10:1970–1979CrossRefPubMedGoogle Scholar
  2. 2.
    Lundstrom K (2006) Structural genomics for membrane proteins. Cell Mol Life Sci 63:2597–2607CrossRefPubMedGoogle Scholar
  3. 3.
    Palczewski K, Kumasaka T, Hori T et al (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745CrossRefPubMedGoogle Scholar
  4. 4.
    Stock D, Leslie AG, Walker JE (1999) Molecular architecture of the rotary motor in ATP synthase. Science 286:1700–1705CrossRefPubMedGoogle Scholar
  5. 5.
    Toyoshima C, Nakasako M, Nomura H, Ogawa H (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405:647–655CrossRefPubMedGoogle Scholar
  6. 6.
    Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346:967–989CrossRefPubMedGoogle Scholar
  7. 7.
    Jidenko M, Nielsen RC, Sorensen TL et al (2005) Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 102:11687–11691CrossRefPubMedGoogle Scholar
  8. 8.
    Long SB, Campbell EB, Mackinnon R (2005) Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309:897–903CrossRefPubMedGoogle Scholar
  9. 9.
    Junge F, Schneider B, Reckel S, Schwarz D, Dotsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol Life Sci 65:1729–1755CrossRefPubMedGoogle Scholar
  10. 10.
    Ago H, Kanaoka Y, Irikura D et al (2007) Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis. Nature 448:609–612CrossRefPubMedGoogle Scholar
  11. 11.
    Cherezov V, Rosenbaum DM, Hanson MA et al (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318:1258–1265CrossRefPubMedGoogle Scholar
  12. 12.
    Ferguson AD, McKeever BM, Xu S et al (2007) Crystal structure of inhibitor-bound human 5-lipoxygenase-activating protein. Science 317:510–512CrossRefPubMedGoogle Scholar
  13. 13.
    Hiroaki Y, Tani K, Kamegawa A et al (2006) Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol 355:628–639CrossRefPubMedGoogle Scholar
  14. 14.
    Jasti J, Furukawa H, Gonzales EB, Gouaux E (2007) Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. Nature 449:316–323CrossRefPubMedGoogle Scholar
  15. 15.
    Long SB, Tao X, Campbell EB, MacKinnon R (2007) Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450:376–382CrossRefPubMedGoogle Scholar
  16. 16.
    Martinez Molina D, Wetterholm A, Kohl A et al (2007) Structural basis for synthesis of inflammatory mediators by human leukotriene C4 synthase. Nature 448:613–616CrossRefPubMedGoogle Scholar
  17. 17.
    Pedersen BP, Buch-Pedersen MJ, Morth JP, Palmgren MG, Nissen P (2007) Crystal structure of the plasma membrane proton pump. Nature 450:1111–1114CrossRefPubMedGoogle Scholar
  18. 18.
    Rasmussen SG, Choi HJ, Rosenbaum DM et al (2007) Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450:383–387CrossRefPubMedGoogle Scholar
  19. 19.
    Standfuss J, Xie G, Edwards PC, Burghammer M, Oprian DD, Schertler GF (2007) Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol 372:1179–1188CrossRefPubMedGoogle Scholar
  20. 20.
    Tornroth-Horsefield S, Gourdon P, Horsefield R et al (2007) Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist. Structure 15:1663–1673CrossRefPubMedGoogle Scholar
  21. 21.
    Warne T, Serrano-Vega MJ, Baker JG et al (2008) Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454:486–491CrossRefPubMedGoogle Scholar
  22. 22.
    Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074CrossRefPubMedGoogle Scholar
  23. 23.
    Geertsma ER, Groeneveld M, Slotboom DJ, Poolman B (2008) Quality control of overexpressed membrane proteins. Proc Natl Acad Sci U S A 105:5722–5727CrossRefPubMedGoogle Scholar
  24. 24.
    Margreiter G, Schwanninger M, Bayer K, Obinger C (2008) Impact of different cultivation and induction regimes on the structure of cytosolic inclusion bodies of TEM1-beta-lactamase. Biotechnol J 3:1245–1255CrossRefPubMedGoogle Scholar
  25. 25.
    Rogl H, Kosemund K, Kuhlbrandt W, Collinson I (1998) Refolding of Escherichia coli produced membrane protein inclusion bodies immobilised by nickel chelating chromatography. FEBS Lett 432:21–26CrossRefPubMedGoogle Scholar
  26. 26.
    Gorzelle BM, Nagy JK, Oxenoid K, Lonzer WL, Cafiso DS, Sanders CR (1999) Reconstitu-tive refolding of diacylglycerol kinase, an integral membrane protein. Biochemistry 38:16373–16382CrossRefPubMedGoogle Scholar
  27. 27.
    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 329:801–814CrossRefPubMedGoogle Scholar
  28. 28.
    Damian M, Perino S, Polidori A et al (2007) New tensio-active molecules stabilize a human G protein-coupled receptor in solution. FEBS Lett 581:1944–1950CrossRefPubMedGoogle Scholar
  29. 29.
    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 260:289–298CrossRefPubMedGoogle Scholar
  30. 30.
    Arechaga I, Miroux B, Karrasch S et al (2000) Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase. FEBS Lett 482:215–219CrossRefPubMedGoogle Scholar
  31. 31.
    Gasson MJ, de Vos WM (ed.) (1994) Genetic and Biotechnology of Lactic acid ­bacteria. Blackie Academic and Professional, London, UK.Google Scholar
  32. 32.
    Teuber M, Geis A (2006) The genus Lactococcus, in the prokaryotes (Dworkin M, ed.), Springer-Verlag, New York 4:205–228Google Scholar
  33. 33.
    Wood BJB, Warner PJ (eds.) (2003) Genetics of lactic acid bacteria. Springer, New YorkGoogle Scholar
  34. 34.
    Monne M, Chan KW, Slotboom DJ, Kunji ER (2005) Functional expression of eukaryotic membrane proteins in Lactococcus lactis. Protein Sci 14:3048–3056CrossRefPubMedGoogle Scholar
  35. 35.
    Aricescu AR, Assenberg R, Bill RM et al (2006) Eukaryotic expression: developments for structural proteomics. Acta Crystallogr D Biol Crystallogr 62:1114–1124CrossRefPubMedGoogle Scholar
  36. 36.
    Bill RM (2001) Yeast—a panacea for the structure-function analysis of membrane proteins? Curr Genet 40:157–171CrossRefPubMedGoogle Scholar
  37. 37.
    Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73:1019–1049CrossRefPubMedGoogle Scholar
  38. 38.
    Lee AG (2004) How lipids affect the activities of integral membrane proteins. Biochim Biophys Acta 1666:62–87CrossRefPubMedGoogle Scholar
  39. 39.
    Hamilton SR, Davidson RC, Sethuraman N et al (2006) Humanization of yeast to produce complex terminally sialylated glycoproteins. Science 313:1441–1443CrossRefPubMedGoogle Scholar
  40. 40.
    Figler RA, Omote H, Nakamoto RK, Al-Shawi MK (2000) Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. Arch Biochem Biophys 376:34–46CrossRefPubMedGoogle Scholar
  41. 41.
    Yelin R, Schuldiner S (2001) Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding. Biochim Biophys Acta 1510:426–441CrossRefPubMedGoogle Scholar
  42. 42.
    De Rivoyre M, Bonino F, Ruel L, Bidet M, Therond P, Mus-Veteau I (2005) Human receptor Smoothened, a mediator of Hedgehog signalling, expressed in its native conformation in yeast. FEBS Lett 579:1529–1533CrossRefPubMedGoogle Scholar
  43. 43.
    Palanivelu DV, Kozono DE, Engel A et al (2006) Co-axial association of recombinant eye lens aquaporin-0 observed in loosely packed 3D crystals. J Mol Biol 355:605–611CrossRefPubMedGoogle Scholar
  44. 44.
    Lenoir G, Menguy T, Corre F et al (2002) Overproduction in yeast and rapid and efficient purification of the rabbit SERCA1a Ca(2+)-ATPase. Biochim Biophys Acta 1560:67–83CrossRefPubMedGoogle Scholar
  45. 45.
    Pisani DF, Rivoyre MD, Ruel L et al (2005) Mouse myodulin, a new potential angiogenic factor, functionally expressed in yeast. Biochem Biophys Res Commun 331:552–556CrossRefPubMedGoogle Scholar
  46. 46.
    Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66CrossRefPubMedGoogle Scholar
  47. 47.
    Nyblom M, Oberg F, Lindkvist-Petersson K et al (2007) Exceptional overproduction of a functional human membrane protein. Protein Expr Purif 56:110–120CrossRefPubMedGoogle Scholar
  48. 48.
    Andre N, Cherouati N, Prual C et al (2006) Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Sci 15:1115–1126CrossRefPubMedGoogle Scholar
  49. 49.
    Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575CrossRefPubMedGoogle Scholar
  50. 50.
    Luckow VA, Summers MD (1988) Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. Virology 167:56–71CrossRefPubMedGoogle Scholar
  51. 51.
    Akermoun M, Koglin M, Zvalova-Iooss D, Folschweiller N, Dowell SJ, Gearing KL (2005) Characterization of 16 human G protein-coupled receptors expressed in baculovirus-infected insect cells. Protein Expr Purif 44:65–74CrossRefPubMedGoogle Scholar
  52. 52.
    Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27:353–365PubMedGoogle Scholar
  53. 53.
    Sondergaard L (1996) Efficiency of different lipofection agents in Drosophila S-2 cells. In Vitro Cell Dev Biol Anim 32:386–387CrossRefPubMedGoogle Scholar
  54. 54.
    Benting J, Lecat S, Zacchetti D, Simons K (2000) Protein expression in Drosophila Schneider cells. Anal Biochem 278:59–68CrossRefPubMedGoogle Scholar
  55. 55.
    Brillet K, da Conceicao MM, Pattus F, Pereira CA (2006) Bioprocess parameters of cell growth and human mu opioid receptor expression in recombinant Drosophila S2 cell cultures in a bioreactor. Bioprocess Biosyst Eng 28:291–293CrossRefPubMedGoogle Scholar
  56. 56.
    Brillet K, Perret BG, Klein V, Pattus F, Wagner R (2008) Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells. Cytotechnology 57:101–109CrossRefPubMedGoogle Scholar
  57. 57.
    De Rivoyre M, Ruel L, Varjosalo M et al (2006) Human receptors patched and smoothened partially transduce hedgehog signal when expressed in Drosophila Cells. J Biol Chem 281:28584–28595CrossRefPubMedGoogle Scholar
  58. 58.
    Perret BG, Wagner R, Lecat S et al (2003) Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors. Protein Expr Purif 31:123–132CrossRefPubMedGoogle Scholar
  59. 59.
    Schetz JA, Kim OJ, Sibley DR (2003) Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells. J Recept Signal Transduct Res 23:99–109CrossRefPubMedGoogle Scholar
  60. 60.
    Zuker CS (1996) The biology of vision of Drosophila. Proc Natl Acad Sci U S A 93:571–576CrossRefPubMedGoogle Scholar
  61. 61.
    Eroglu C, Cronet P, Panneels V, Beaufils P, Sinning I (2002) Functional reconstitution of purified metabotropic glutamate receptor expressed in the fly eye. EMBO Rep 3:491–496CrossRefPubMedGoogle Scholar
  62. 62.
    Sarramegna V, Muller I, Milon A, Talmont F (2006) Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure. Cell Mol Life Sci 63:1149–1164CrossRefGoogle Scholar
  63. 63.
    Eifler N, Duckely M, Sumanovski LT et al (2007) Functional expression of mammalian receptors and membrane channels in different cells. J Struct Biol 159:179–193CrossRefPubMedGoogle Scholar
  64. 64.
    Tate CG, Haase J, Baker C et al (2003) Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter. Biochim Biophys Acta 1610:141–153CrossRefPubMedGoogle Scholar
  65. 65.
    Baldi L, Hacker DL, Adam M, Wurm FM (2007) Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol Lett 29:677–684CrossRefPubMedGoogle Scholar
  66. 66.
    Lundstrom K, Mills A, Buell G, Allet E, Adami N, Liljestrom P (1994) High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur J Biochem 224:917–921CrossRefPubMedGoogle Scholar
  67. 67.
    Hovius R, Tairi AP, Blasey H, Bernard A, Lundstrom K, Vogel H (1998) Characterization of a mouse serotonin 5-HT3 receptor purified from mammalian cells. J Neurochem 70:824–834CrossRefPubMedGoogle Scholar
  68. 68.
    Hassaine G, Wagner R, Kempf J et al (2006) Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells. Protein Expr Purif 45:343–351CrossRefPubMedGoogle Scholar
  69. 69.
    Lundstrom K, Michel A, Blasey H et al (1997) Expression of ligand-gated ion channels with the Semliki forest virus expression system. J Recept Signal Transduct Res 17:115–126CrossRefPubMedGoogle Scholar
  70. 70.
    Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A (2004) Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry 43:12585–12591CrossRefPubMedGoogle Scholar
  71. 71.
    Elbaz Y, Steiner-Mordoch S, Danieli T, Schuldiner S (2004) In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc Natl Acad Sci U S A 101:1519–1524CrossRefPubMedGoogle Scholar
  72. 72.
    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 41:27–37CrossRefPubMedGoogle Scholar
  73. 73.
    Klammt C, Schwarz D, Fendler K, Haase W, Dotsch V, Bernhard F (2005) Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J 272:6024–6038CrossRefPubMedGoogle Scholar
  74. 74.
    Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V, Bernhard F (2006) Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J 273:4141–4153CrossRefPubMedGoogle Scholar
  75. 75.
    Keller T, Schwarz D, Bernhard F et al (2008) Cell free expression and functional reconstitution of eukaryotic drug transporters. Biochemistry 47:4552–4564CrossRefPubMedGoogle Scholar
  76. 76.
    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 158:482–493CrossRefPubMedGoogle Scholar
  77. 77.
    Liguori L, Marques B, Villegas-Mendez A, Rothe R, Lenormand JL (2007) Production of membrane proteins using cell-free expression systems. Expert Rev Proteomics 4:79–90CrossRefPubMedGoogle Scholar
  78. 78.
    Park KH, Berrier C, Lebaupain F et al (2007) Fluorinated and hemifluorinated surfactants as alternatives to detergents for membrane protein cell-free synthesis. Biochem J 403:183–187CrossRefPubMedGoogle Scholar
  79. 79.
    Kalmbach R, Chizhov I, Schumacher MC, Friedrich T, Bamberg E, Engelhard M (2007) Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J Mol Biol 371:639–648CrossRefPubMedGoogle Scholar
  80. 80.
    Liguori L, Marques B, Villegas-Mendez A, Rothe R, Lenormand JL (2008) Liposomes-mediated delivery of pro-apoptotic therapeutic membrane proteins. J Control Rel 126:217–227CrossRefGoogle Scholar
  81. 81.
    Nozawa A, Nanamiya H, Miyata T et al (2007) A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant Cell Physiol 48:1815–1820CrossRefPubMedGoogle Scholar
  82. 82.
    Prive GG (2007) Detergents for the stabilization and crystallization of membrane proteins. Methods 41:388–397CrossRefPubMedGoogle Scholar
  83. 83.
    Polidori A, Presset M, Lebaupain F et al (2006) Fluorinated and hemifluorinated surfactants derived from maltose: synthesis and application to handling membrane proteins in aqueous solution. Bioorg Med Chem Lett 16:5827–5831CrossRefPubMedGoogle Scholar
  84. 84.
    Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci U S A 93:15047–15050CrossRefPubMedGoogle Scholar
  85. 85.
    Breyton C, Chabaud E, Chaudier Y, Pucci B, Popot JL (2004) Hemifluorinated surfactants: a non-dissociating environment for handling membrane proteins in aqueous solutions? FEBS Lett 564:312–318CrossRefPubMedGoogle Scholar
  86. 86.
    Chabaud E, Barthelemy P, Mora N, Popot JL, Pucci B (1998) Stabilization of integral membrane proteins in aqueous solution using fluorinated surfactants. Biochimie 80:515–530CrossRefPubMedGoogle Scholar
  87. 87.
    Tornroth-Horsefield S, Wang Y, Hedfalk K et al (2006) Structural mechanism of plant aquaporin gating. Nature 439:688–694CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Institut of Developmental Biology and Cancer, UMR CNRS 6543, Université de Nice-Sophia Antipolis, Parc ValroseNiceFrance

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