Practical Aspects in Expression and Purification of Membrane Proteins for Structural Analysis

  • Kutti R. Vinothkumar
  • Patricia C. Edwards
  • Joerg Standfuss
Part of the Methods in Molecular Biology book series (MIMB, volume 955)


A surge of membrane protein structures in the last few years can be attributed to advances in technologies starting at the level of genomes, to highly efficient expression systems, stabilizing conformational flexibility, automation of crystallization and data collection for screening large numbers of crystals and the microfocus beam lines at synchrotrons. The substantial medical importance of many membrane proteins provides a strong incentive to understand them at the molecular level. It is becoming obvious that the major bottleneck in many of the membrane projects is obtaining sufficient amount of stable functional proteins in a detergent micelle for structural studies. Naturally, large effort has been spent on optimizing and advancing multiple expression systems and purification strategies that have started to yield sufficient protein and structures. We describe in this chapter protocols to refold membrane proteins from inclusion bodies, purification from inner membranes of Escherichia coli and from mammalian cell lines.

Key words

Expression systems Escherichia coli Inclusion bodies HEK cells Rhodopsin 



VK acknowledges the European Union for a Marie-Curie Intra-European Fellowship and the Medical Research Council for a Career Development fellowship. The work on OmpG was done by VK at the Max-Planck Institute of Biophysics, Frankfurt as part of Ph.D. thesis. This work was further supported by the Swiss National Science Foundation (SNSF) grants 31003A_132815 and 31003A_141235 to JS. We thank Gebhard Schertler at the PSI and Daniel Oprian at the Brandeis University for discussion and advice on the expression of rhodopsin.


  1. 1.
    Vinothkumar KR, Henderson R (2010) Structures of membrane proteins. Q Rev Biophys 43:65–158PubMedCrossRefGoogle Scholar
  2. 2.
    Tate CG, Haase J, Baker C, Boorsma M, Magnani F, Vallis Y, Williams DC (2003) Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter. Biochim Biophys Acta 1610:141–153PubMedCrossRefGoogle Scholar
  3. 3.
    Junge F, Schneider B, Reckel S, Schwarz D, Dotsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol Life Sci 65:1729–1755PubMedCrossRefGoogle Scholar
  4. 4.
    Junge F, Haberstock S, Roos C, Stefer S, Proverbio D, Dotsch V, Bernhard F (2010) Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins. N Biotechnol 28:262–271PubMedCrossRefGoogle Scholar
  5. 5.
    Chen YJ, Pornillos O, Lieu S, Ma C, Chen AP, Chang G (2007) X-ray structure of EmrE supports dual topology model. Proc Natl Acad Sci USA 104:18999–19004PubMedCrossRefGoogle Scholar
  6. 6.
    Sorensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128PubMedCrossRefGoogle Scholar
  7. 7.
    Midgett CR, Madden DR (2007) Breaking the bottleneck: eukaryotic membrane protein expression for high-resolution structural studies. J Struct Biol 160:265–274PubMedCrossRefGoogle Scholar
  8. 8.
    Lee JK, Stroud RM (2010) Unlocking the eukaryotic membrane protein structural proteome. Curr Opin Struct Biol 20:464–470PubMedCrossRefGoogle Scholar
  9. 9.
    Mus-Veteau I (2010) Heterologous expression of membrane proteins for structural analysis. Methods Mol Biol 601:1–16PubMedCrossRefGoogle Scholar
  10. 10.
    Yildiz O, Vinothkumar KR, Goswami P, Kuhlbrandt W (2006) Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation. EMBO J 25:3702–3713PubMedCrossRefGoogle Scholar
  11. 11.
    Wang Y, Zhang Y, Ha Y (2006) Crystal structure of a rhomboid family intramembrane protease. Nature 444:179–180PubMedCrossRefGoogle Scholar
  12. 12.
    Vinothkumar KR (2011) Structure of rhomboid protease in a lipid environment. J Mol Biol 407:232–247PubMedCrossRefGoogle Scholar
  13. 13.
    Standfuss J, Xie G, Edwards PC, Burghammer M, Oprian DD, Schertler GF (2007) Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol 372:1179–1188PubMedCrossRefGoogle Scholar
  14. 14.
    Standfuss J, Edwards PC, D’Antona A, Fransen M, Xie G, Oprian DD, Schertler GF (2011) The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656–660PubMedCrossRefGoogle Scholar
  15. 15.
    Hiller S, Abramson J, Mannella C, Wagner G, Zeth K (2010) The 3D structures of VDAC represent a native conformation. Trends Biochem Sci 35:514–521PubMedCrossRefGoogle Scholar
  16. 16.
    Standfuss J, Kuhlbrandt W (2004) The three isoforms of the light-harvesting complex II: spectroscopic features, trimer formation, and functional roles. J Biol Chem 279:36884–36891PubMedCrossRefGoogle Scholar
  17. 17.
    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 329:801–814PubMedCrossRefGoogle Scholar
  18. 18.
    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 280:20253–20260PubMedCrossRefGoogle Scholar
  19. 19.
    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 128:7402–7403PubMedCrossRefGoogle Scholar
  20. 20.
    Gruswitz F, Chaudhary S, Ho JD, Schlessinger A, Pezeshki B, Ho CM, Sali A, Westhoff CM, Stroud RM (2010) Function of human Rh based on structure of RhCG at 2.1 A. Proc Natl Acad Sci USA 107:9638–9643PubMedCrossRefGoogle Scholar
  21. 21.
    Reeves PJ, Kim JM, Khorana HG (2002) Structure and function in rhodopsin: a tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants. Proc Natl Acad Sci USA 99:13413–13418PubMedCrossRefGoogle Scholar
  22. 22.
    Reeves PJ, Callewaert N, Contreras R, Khorana HG (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylgluco­saminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci USA 99:13419–13424PubMedCrossRefGoogle Scholar
  23. 23.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  24. 24.
    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 260:289–298PubMedCrossRefGoogle Scholar
  25. 25.
    Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83:346–356PubMedCrossRefGoogle Scholar
  26. 26.
    Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI, Dwek RA, Jones EY, Owens RJ, Davis SJ (2007) Glycoprotein structural genomics: solving the glycosylation problem. Structure 15:267–273PubMedCrossRefGoogle Scholar
  27. 27.
    Deupi X, Edwards P, Singhal A, Nickle B, Oprian D, Schertler G, Standfuss J (2012) Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II 109:119–124PubMedCrossRefGoogle Scholar
  28. 28.
    Zoonens M, Miroux B (2010) Expression of membrane proteins at the Escherichia coli membrane for structural studies. Methods Mol Biol 601:49–66PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kutti R. Vinothkumar
    • 1
  • Patricia C. Edwards
    • 1
  • Joerg Standfuss
    • 2
  1. 1.MRC Laboratory of Molecular BiologyCambridgeUK
  2. 2.Laboratory of Biomolecular ResearchPaul Scherrer InstituteVilligenSwitzerland

Personalised recommendations