Skip to main content
SpringerLink
Log in

Production of Membrane Proteins in Escherichia coli and Lactococcus lactis

  • Protocol
  • First Online:
Heterologous Expression of Membrane Proteins

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 601))

Abstract

As the equivalent to gatekeepers of the cell, membrane transport proteins perform a variety of critical functions. Progress on the functional and structural characterization of membrane proteins is slowed due to problems associated with their (heterologous) overexpression. Often, overexpression fails or leads to aggregated material from which the production of functionally refolded protein is challenging. It is still difficult to predict whether a given membrane protein can be overproduced in a functional competent state. As a result, the most straightforward strategy to set up an overexpression system is to screen a multitude of conditions, including the comparison of homologues, type and location of (affinity) tags, and distinct expression hosts. Here, we detail methodology to rapidly establish and optimize (membrane) protein expression in Escherichia coli and Lactococcus lactis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Driessen AJ, Nouwen N (2008) Protein translocation across the bacterial cytoplasmic membrane. Annu Rev Biochem 77:643–667

    Article  CAS  PubMed  Google Scholar 

  2. Korepanova A, Gao FP, Hua Y, Qin H, Nakamoto RK, Cross TA (2005) Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli. Protein Sci 14:148–158

    Article  CAS  PubMed  Google Scholar 

  3. Dobrovetsky E, Lu ML, Andorn-Broza R, Khutoreskaya G, Bray JE, Savchenko A, Arrowsmith CH, Edwards AM, Koth CM (2005) High-throughput production of prokaryotic membrane proteins. J Struct Funct Genom 6:33–50

    Article  CAS  Google Scholar 

  4. White MA, Clark KM, Grayhack EJ, Dumont ME (2007) Characteristics affecting expression and solubilization of yeast membrane proteins. J Mol Biol 365:621–636

    Article  CAS  PubMed  Google Scholar 

  5. Wagner S, Baars L, Ytterberg AJ, Klussmeier A, Wagner CS, Nord O, Nygren PA, van Wijk KJ, de Gier JW (2007) Consequences of membrane protein overexpression in Escherichia coli. Mol Cell Proteom 6:1527–1550

    Article  CAS  Google Scholar 

  6. Bonander N, Hedfalk K, Larsson C, Mostad P, Chang C, Gustafsson L, Bill RM (2005) Design of improved membrane protein production experiments: quantitation of the host response. Protein Sci 14:1729–1740

    Article  CAS  PubMed  Google Scholar 

  7. Savchenko A, Yee A, Khachatryan A, Skarina T, Evdokimova E, Pavlova M, Semesi A, Northey J, Beasley S, Lan N, Das R, Gerstein M, Arrowmith CH, Edwards AM (2003) Strategies for structural proteomics of prokaryotes: quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches. Proteins 50:392–399

    Article  CAS  PubMed  Google Scholar 

  8. Locher KP, Lee AT, Rees DC (2002) The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296:1091–1098

    Article  CAS  PubMed  Google Scholar 

  9. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533

    CAS  PubMed  Google Scholar 

  10. Surade S, Klein M, Stolt-Bergner PC, Muenke C, Roy A, Michel H (2006) Comparative analysis and “expression space” coverage of the production of prokaryotic membrane proteins for structural genomics. Protein Sci 15:2178–2189

    Article  CAS  PubMed  Google Scholar 

  11. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211–222

    Article  CAS  PubMed  Google Scholar 

  12. Grisshammer R, Tate CG (1995) Overexpres­sion of integral membrane proteins for structural studies. Q Rev Biophys 28:315–422

    Article  CAS  PubMed  Google Scholar 

  13. Kunji ER, Slotboom DJ, Poolman B (2003) Lactococcus lactis as host for overproduction of functional membrane proteins. Biochim Biophys Acta 1610:97–108

    Article  CAS  PubMed  Google Scholar 

  14. Quick M, Javitch JA (2007) Monitoring the function of membrane transport proteins in detergent-solubilized form. Proc Natl Acad Sci USA 104:3603–3608

    Article  CAS  PubMed  Google Scholar 

  15. de Ruyter PG, Kuipers OP, de Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667

    PubMed  Google Scholar 

  16. Geertsma ER, Poolman B (2007) High-throughput cloning and expression in recalcitrant bacteria. Nat Methods 4:705–707

    Article  CAS  PubMed  Google Scholar 

  17. Poolman B, Konings WN (1988) Relation of growth of Streptococcus lactis and Streptococcus cremoris to amino acid transport. J Bacteriol 170:700–707

    CAS  PubMed  Google Scholar 

  18. Wegmann U, O’Connell-Motherway M, Zomer A, Buist G, Shearman C, Canchaya C, Ventura M, Goesmann A, Gasson MJ, Kuipers OP, van Sinderen D, Kok J (2007) Complete genome sequence of the prototype lactic acid bacterium Lactococcus lactis subsp. cremoris MG1363. J Bacteriol 189:3256–3270

    Article  CAS  PubMed  Google Scholar 

  19. Nouaille S, Morello E, Cortez-Peres N, Le Loir Y, Commissaire J, Gratadoux JJ, Poumerol E, Gruss A, Langella P (2006) Complementation of the Lactococcus lactis secretion machinery with Bacillus subtilis SecDF improves secretion of staphylococcal nuclease. Appl Environ Microbiol 72:2272–2279

    Article  CAS  PubMed  Google Scholar 

  20. van der Sluis EO, Driessen AJ (2006) Stepwise evolution of the Sec machinery in Proteobacteria. Trends Microbiol 14:105–108

    Article  PubMed  Google Scholar 

  21. Saaf A, Monne M, de Gier JW, von Heijne G (1998) Membrane topology of the 60-kDa Oxa1p homologue from Escherichia coli. J Biol Chem 273:30415–30418

    Article  CAS  PubMed  Google Scholar 

  22. Driessen AJ, Zheng T, In’t Veld G, Op den Kamp JA, Konings WN (1988) Lipid requirement of the branched-chain amino acid ­transport system of Streptococcus cremoris. Biochemistry 27:865–872

    Article  CAS  PubMed  Google Scholar 

  23. Nakamura Y, Gojobori T, Ikemura T (2000) Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res 28:292

    Article  CAS  PubMed  Google Scholar 

  24. Kouwen TR, van der Goot A, Dorenbos R, Winter T, Antelmann H, Plaisier MC, Quax WJ, van Dijl JM, Dubois JY (2007) Thiol-disulphide oxidoreductase modules in the low-GC Gram-positive bacteria. Mol Microbiol 64:984–999

    Article  CAS  PubMed  Google Scholar 

  25. Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289:1352–1355

    Article  CAS  PubMed  Google Scholar 

  26. Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Vermeire A, Goddeeris B, Cox E, Remon JP, Remaut E (2003) Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 21:785–789

    Article  CAS  PubMed  Google Scholar 

  27. Vandenbroucke K, Hans W, Van Huysse J, Neirynck S, Demetter P, Remaut E, Rottiers P, Steidler L (2004) Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology 127:502–513

    Article  CAS  PubMed  Google Scholar 

  28. Kunji ER, Chan KW, Slotboom DJ, Floyd S, O’Connor R, Monne M (2005) Eukaryotic membrane protein overproduction in Lacto­coccus lactis. Curr Opin Biotechnol 16:546–551

    Article  CAS  PubMed  Google Scholar 

  29. Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074

    Article  CAS  PubMed  Google Scholar 

  30. Walhout AJ, Temple GF, Brasch MA, Hartley JL, Lorson MA, van den Heuvel S, Vidal M (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328:575–592

    Article  CAS  PubMed  Google Scholar 

  31. Liu Q, Li MZ, Leibham D, Cortez D, Elledge SJ (1998) The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes. Curr Biol 8:1300–1309

    Article  CAS  PubMed  Google Scholar 

  32. 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 177:4121–4130

    CAS  PubMed  Google Scholar 

  33. Wang DN, Safferling M, Lemieux MJ, Griffith H, Chen Y, Li XD (2003) Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim Biophys Acta 1610:23–36

    Article  CAS  PubMed  Google Scholar 

  34. Chen Y, Song J, Sui SF, Wang DN (2003) DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Protein Exp Purif 32:221–231

    Article  CAS  Google Scholar 

  35. Waldo GS, Standish BM, Berendzen J, Terwilliger TC (1999) Rapid protein-folding assay using green fluorescent protein. Nat Biotechnol 17:691–695

    Article  CAS  PubMed  Google Scholar 

  36. Drew DE, von Heijne G, Nordlund P, de Gier JW (2001) Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli. FEBS Lett 507:220–224

    Article  CAS  PubMed  Google Scholar 

  37. Drew D, Lerch M, Kunji E, Slotboom DJ, de Gier JW (2006) Optimization of membrane protein overexpression and purification using GFP fusions. Nat Methods 3:303–313

    Article  CAS  PubMed  Google Scholar 

  38. Geertsma ER, Groeneveld M, Slotboom DJ, Poolman B (2008) Quality control of overexpressed membrane proteins. Proc Natl Acad Sci USA 105:5722–5727

    Article  CAS  PubMed  Google Scholar 

  39. Kawate T, Gouaux E (2006) Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14:673–681

    Article  CAS  PubMed  Google Scholar 

  40. Sambrook J, Russell DW (2001) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  41. Terzaghi BE, Sandine WE (1975) Improved medium for lactic Streptococci and their bacteriophages. Appl Microbiol 29:807–813

    CAS  PubMed  Google Scholar 

  42. Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991) “Touchdown” PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19:4008

    Article  CAS  PubMed  Google Scholar 

  43. Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lacto­coccus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55: 3119–3123

    CAS  PubMed  Google Scholar 

  44. Berntsson RP, Alia Oktaviani N, Fusetti F, Thunnissen AM, Poolman B, Slotboom DJ (2009) Selenomethionine incorporation in proteins expressed in Lactococcus lactis. Protein Sci 18(5):1121–1127

    Article  CAS  PubMed  Google Scholar 

  45. El Khattabi M, van Roosmalen ML, Jager D, Metselaar H, Permentier H, Leenhouts K, Broos J (2008) Lactococcus lactis as expression host for the biosynthetic incorporation of tryptophan analogues into recombinant proteins. Biochem J 409(1): 193–198

    Article  Google Scholar 

Download references

Acknowledgments

We thank G.K. Schuurman-Wolters, M.B. Tol, M. Groeneveld, D.J. Slotboom, and S. Henstra for their contribution in establishing the methodologies discussed. This work was funded by the European Membrane Protein Consortium (grant 504601), a FEBS fellowship (to E.R.G.), and the Netherlands Proteomics Centre.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Netherlands Proteomics Centre (NPC), and Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands

    Eric R. Geertsma & Bert Poolman

Authors
  1. Eric R. Geertsma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bert Poolman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bert Poolman .

Editor information

Editors and Affiliations

  1. CNRS - UMR 6543, Université Nice, parc Valrose, Nice CX 02, 06108, France

    Isabelle Mus-Veteau

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Geertsma, E.R., Poolman, B. (2010). Production of Membrane Proteins in Escherichia coli and Lactococcus lactis . 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_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-344-2_2

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-343-5

  • Online ISBN: 978-1-60761-344-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation