Membrane Protein Production in Escherichia coli: Overview and Protocols

  • Georges Hattab
  • Annabelle Y. T. Suisse
  • Oana Ilioaia
  • Marina Casiraghi
  • Manuela Dezi
  • Xavier L. Warnet
  • Dror E. Warschawski
  • Karine Moncoq
  • Manuela Zoonens
  • Bruno Miroux


Structural biology of membrane proteins is hampered by the difficulty to express and purify them in a large amount. Despite recent progress in biophysical methods that have reduced the need of biological materials, membrane protein production remains a bottleneck in the field and will require further conceptual and technological developments. Among the unique 424 membrane protein structures found in protein databases, about half of them come from proteins produced in Escherichia coli. In this chapter, we have reviewed the existing bacterial expression systems. The T7 RNA polymerase-based expression system accounts for up to 62 % of solved heterologous membrane protein structures. Among the dozen of bacterial hosts available, the mutant hosts C41(DE3) and C43(DE3) have contributed to half of the integral membrane protein structures that were solved after production using the T7 expression system. After a general introduction on this expression system, the protocol section of this chapter provides detailed protocols to select bacterial expression mutant hosts and to optimize culture conditions.


Structural biology Membrane protein Recombinant expression Escherichia coli T7 RNA polymerase Bibliographic analysis 



This work was supported by the Agence National de La Recherche (ANR MIT-2M, 2010 BLAN1518), the Centre National de la Recherche Scientifique, and by the “Initiative d’Excellence” programme from the French State (Grant “DYNAMO”, ANR-11-LABEX-0011–01).


  1. Abdine A, Verhoeven MA, Park K-H et al (2010) Structural study of the membrane protein MscL using cell-free expression and solid-state NMR. J Magn Reson 204:155–159. doi:10.1016/j.jmr.2010.02.003PubMedCrossRefGoogle Scholar
  2. Alfasi S, Sevastsyanovich Y, Zaffaroni L et al (2011) Use of GFP fusions for the isolation of Escherichia coli strains for improved production of different target recombinant proteins. J Biotechnol 156:11–21. doi:10.1016/j.jbiotec.2011.08.016PubMedCrossRefGoogle Scholar
  3. Alkhalfioui F, Logez C, Bornert O, Wagner R (2011) In: Robinson AS (ed) Production of membrane proteins: strategies for expression and isolation. Wiley-VCH, Weinheim, pp 75–108Google Scholar
  4. 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 F1F0 ATP synthase. FEBS Lett 482:215–219PubMedCrossRefGoogle Scholar
  5. Arechaga I, Miroux B, Runswick MJ, Walker JE (2003) Over-expression of Escherichia coli F1F0–ATPase subunit a is inhibited by instability of the uncB gene transcript. FEBS Lett 547:97–100PubMedCrossRefGoogle Scholar
  6. Banères J-L, Popot J-L, Mouillac B (2011) New advances in production and functional folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322. doi:10.1016/j.tibtech.2011.03.002PubMedCrossRefGoogle Scholar
  7. Bocquet N, Nury H, Baaden M et al (2008) X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 457:111–114. doi:10.1038/nature07462PubMedCrossRefGoogle Scholar
  8. Catoire LJ, Damian M, Giusti F et al (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 132:9049–9057. doi:10.1021/ja101868cPubMedCrossRefGoogle Scholar
  9. Chae PS, Rasmussen SGF, Rana RR et al (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat Methods 7:1003–1008. doi:10.1038/nmeth.1526PubMedCentralPubMedCrossRefGoogle Scholar
  10. Chen Y, Song J, Sui S, Wang D-N (2003) DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Protein Expr Purif 32:221–231. doi:10.1016/S1046-5928(03)00233-XPubMedCrossRefGoogle Scholar
  11. Dong H, Nilsson L, Kurland CG (1995) Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction. J Bacteriol 177:1497–1504PubMedCentralPubMedGoogle Scholar
  12. Drew D, Lerch M, Kunji E et al (2006) Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3:303–313. doi:10.1038/nmeth0406-303PubMedCrossRefGoogle Scholar
  13. Eriksson HM, Wessman P, Ge C et al (2009) Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase. J Biol Chem 284:33904–33914. doi:10.1074/jbc.M109.021618PubMedCentralPubMedCrossRefGoogle Scholar
  14. Fairman JW, Dautin N, Wojtowicz D et al (2012) Crystal structures of the outer membrane domain of intimin and invasin from enterohemorrhagic E. coli and enteropathogenic Y. pseudotuberculosis. Structure 20:1233–1243. doi:10.1016/j.str.2012.04.011PubMedCentralPubMedCrossRefGoogle Scholar
  15. Frelet-Barrand A, Boutigny S, Kunji ERS, Rolland N (2010) Membrane protein expression in Lactococcus lactis. Methods Mol Biol 601:67–85. doi:10.1007/978-1-60761-344-2_5PubMedCrossRefGoogle Scholar
  16. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130PubMedCentralPubMedGoogle Scholar
  17. Hattab G, Moncoq K, Warschawski DE, Miroux B (2014) Escherichia coli as host for membrane protein structure determination: a global analysis. Biophys J 106(2, Suppl 1):46aGoogle Scholar
  18. Jidenko M, Nielsen RC, Sørensen TL-M et al (2005) Crystallization of a mammalian membrane protein overexpressed in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 102:11687–11691. doi:10.1073/pnas.0503986102PubMedCentralPubMedCrossRefGoogle Scholar
  19. Miot M, Betton J-M (2011) Reconstitution of the Cpx signaling system from cell-free synthesized proteins. New Biotechnol 28:277–281. doi:10.1016/j.nbt.2010.06.012CrossRefGoogle Scholar
  20. 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 260:289–298. doi:10.1006/jmbi.1996.0399PubMedCrossRefGoogle Scholar
  21. Miroux B, Frossard V, Raimbault S et al (1993) The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins. EMBO J 12:3739–3745PubMedCentralPubMedGoogle Scholar
  22. Moffatt BA, Studier FW (1987) T7 lysozyme inhibits transcription by T7 RNA polymerase. Cell 49:221–227PubMedCrossRefGoogle Scholar
  23. Mouillac B, Banères J-L (2010) Mammalian membrane receptors expression as inclusion bodies in Escherichia coli. Methods Mol Biol 601:39–48. doi:10.1007/978-1-60761-344-2_3PubMedCrossRefGoogle Scholar
  24. Nørholm MHH, Toddo S, Virkki MTI et al (2013) Improved production of membrane proteins in Escherichia coli by selective codon substitutions. FEBS Lett 587:2352–2358. doi:10.1016/j.febslet.2013.05.063PubMedCrossRefGoogle Scholar
  25. Nury H, Renterghem CV, Weng Y et al (2011) X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. Nature 469:428–431. doi:10.1038/nature09647PubMedCrossRefGoogle Scholar
  26. Oldham RK, Dillman RO (2008) Monoclonal antibodies in cancer therapy: 25 years of progress. J Clin Oncol 26:1774–1777. doi:10.1200/JCO.2007.15.7438PubMedCrossRefGoogle Scholar
  27. Orriss GL, Runswick MJ, Collinson IR et al (1996) The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex. Biochem J 314(Pt 2):695–700PubMedCentralPubMedGoogle Scholar
  28. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996. doi:10.1038/nrd2199PubMedCrossRefGoogle Scholar
  29. Park SH, Das BB, Casagrande F et al (2012) Structure of the chemokine receptor CXCR1 in phospholipid bilayers. Nature 491:779–783. doi:10.1038/nature11580PubMedCentralPubMedCrossRefGoogle Scholar
  30. Pechmann S, Frydman J (2013) Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding. Nat Struct Mol Biol 20:237–243. doi:10.1038/nsmb.2466PubMedCentralPubMedCrossRefGoogle Scholar
  31. Popot J-L, Althoff T, Bagnard D et al (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408. doi:10.1146/annurev-biophys-042910-155219PubMedCrossRefGoogle Scholar
  32. Rogé J, Betton J-M (2005) Use of pIVEX plasmids for protein overproduction in Escherichia coli. Microb Cell Fact 4:18. doi:10.1186/1475-2859-4-18PubMedCentralPubMedCrossRefGoogle Scholar
  33. Sarkar CA, Dodevski I, Kenig M et al (2008) From the cover: directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc Natl Acad Sci U S A 105:14808–14813. doi:10.1073/pnas.0803103105PubMedCentralPubMedCrossRefGoogle Scholar
  34. Sevastsyanovich YR, Alfasi SN, Cole JA (2010) Sense and nonsense from a systems biology approach to microbial recombinant protein production. Biotechnol Appl Biochem 55:9–28. doi:10.1042/BA20090174PubMedCrossRefGoogle Scholar
  35. Shaw AZ, Miroux B (2003) A general approach for heterologous membrane protein expression in Escherichia coli: the uncoupling protein, UCP1, as an example. Methods Mol Biol 228:23–35. doi:10.1385/1-59259-400-X:23PubMedGoogle Scholar
  36. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89PubMedCrossRefGoogle Scholar
  37. Supply P, Wach A, Thinès-Sempoux D, Goffeau A (1993) Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae. J Biol Chem 268:19744–19752PubMedGoogle Scholar
  38. Tate CG (2012) A crystal clear solution for determining G-protein-coupled receptor structures. Trends Biochem Sci 37:343–352. doi:10.1016/j.tibs.2012.06.003PubMedCrossRefGoogle Scholar
  39. Tifrea DF, Sun G, Pal S et al (2011) Amphipols stabilize the Chlamydia major outer membrane protein and enhance its protective ability as a vaccine. Vaccine 29:4623–4631. doi:10.1016/j.vaccine.2011.04.065PubMedCentralPubMedCrossRefGoogle Scholar
  40. Von Meyenburg K, Jørgensen BB, Van Deurs B (1984) Physiological and morphological effects of overproduction of membrane-bound ATP synthase in Escherichia coli K-12. EMBO J 3:1791–1797PubMedCentralPubMedGoogle Scholar
  41. Wagner S, Baars L, Ytterberg AJ et al (2007) Consequences of membrane protein overexpression in Escherichia coli. Mol Cell Proteomics 6:1527–1550. doi:10.1074/mcp.M600431-MCP200PubMedCrossRefGoogle Scholar
  42. Wagner S, Klepsch MM, Schlegel S et al (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci U S A 105:14371–14376. doi:10.1073/pnas.0804090105PubMedCentralPubMedCrossRefGoogle Scholar
  43. Walker J, Miroux B (1999) Selection of Escherichia coli hosts that are optimized for the overexpression of proteins. In: Demain AL, Davies JE (eds) Manual of industrial microbiology and biotechnology (MIMB2), 2nd edn. ASM, Washington DCGoogle Scholar
  44. Walse B, Dufe VT, Svensson B et al (2008) The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites. Biochemistry 47:8929–8936. doi:10.1021/bi8003318PubMedCrossRefGoogle Scholar
  45. Warschawski DE (2013) Membrane proteins of known structure determined by NMR. Accessed 30 Aug 2013
  46. Way M, Pope B, Gooch J et al (1990) Identification of a region in segment 1 of gelsolin critical for actin binding. EMBO J 9:4103–4109PubMedCentralPubMedGoogle Scholar
  47. Weiner JH, Lemire BD, Elmes ML et al (1984) Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle. J Bacteriol 158:590–596PubMedCentralPubMedGoogle Scholar
  48. White S (2013) Membrane proteins of known 3D structure determined by X-ray crystallography. Accessed 30 Aug 2013
  49. Wilkison WO, Walsh JP, Corless JM, Bell RM (1986) Crystalline arrays of the Escherichia coli sn-glycerol-3-phosphate acyltransferase, an integral membrane protein. J Biol Chem 261:9951–9958PubMedGoogle Scholar
  50. Wright R, Basson M, D’Ari L, Rine J (1988) Increased amounts of HMG-CoA reductase induce “karmellae”: a proliferation of stacked membrane pairs surrounding the yeast nucleus. J Cell Biol 107:101–114PubMedCrossRefGoogle Scholar
  51. Zoonens M, Miroux B (2010) Expression of membrane proteins at the Escherichia coli membrane for structural studies. Methods Mol Biol 601:49–66. doi:10.1007/978-1-60761-344-2_4PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Georges Hattab
    • 1
  • Annabelle Y. T. Suisse
    • 1
  • Oana Ilioaia
    • 1
  • Marina Casiraghi
    • 1
  • Manuela Dezi
    • 2
  • Xavier L. Warnet
    • 1
  • Dror E. Warschawski
    • 1
  • Karine Moncoq
    • 1
  • Manuela Zoonens
    • 1
  • Bruno Miroux
    • 1
  1. 1.Laboratory of Physico-Chemical Biology of Membrane Proteins, UMR-CNRS 7099Institute of Physico-Chemical Biology, and Université Paris DiderotParisFrance
  2. 2.Laboratory of Crystallography and NMR Biology, UMR-CNRS 8015Université Paris DescartesParisFrance

Personalised recommendations