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Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions

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Membrane Biogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1033))

Abstract

Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purification is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the production of high-quality membrane protein material for functional and structural studies.

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References

  1. Wagner S, Bader ML, Drew D, de Gier JW (2006) Rationalizing membrane protein overexpression. Trends Biotechnol 24:364–371

    Article  PubMed  CAS  Google Scholar 

  2. Samuelson JC (2011) Recent developments in difficult protein expression: a guide to E. coli strains, promoters, and relevant host mutations. Methods Mol Biol 705:195–209

    Article  PubMed  CAS  Google Scholar 

  3. Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M, Hogbom M, van Wijk KJ, Slotboom DJ, Persson JO, de Gier JW (2008) Tuning Escherichia coli for membrane protein overexpression. Proc Natl Acad Sci USA 105:14371–14376

    Article  PubMed  CAS  Google Scholar 

  4. Schlegel S, Lofblom J, Lee C, Hjelm A, Klepsch M, Strous M, Drew D, Slotboom DJ, de Gier JW (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J Mol Biol 423(4):648–659

    Article  PubMed  CAS  Google Scholar 

  5. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130

    Article  PubMed  CAS  Google Scholar 

  6. Giacalone MJ, Gentile AM, Lovitt BT, Berkley NL, Gunderson CW, Surber MW (2006) Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques 40:355–364

    Article  PubMed  CAS  Google Scholar 

  7. 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 Proteomics 6:1527–1550

    Article  PubMed  CAS  Google Scholar 

  8. 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  PubMed  CAS  Google Scholar 

  9. Drew D, Slotboom DJ, Friso G, Reda T, Genevaux P, Rapp M, Meindl-Beinker NM, Lambert W, Lerch M, Daley DO, Van Wijk KJ, Hirst J, Kunji E, De Gier JW (2005) A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci 14:2011–2017

    Article  PubMed  CAS  Google Scholar 

  10. Drew D, Sjostrand D, Nilsson J, Urbig T, Chin CN, de Gier JW, von Heijne G (2002) Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis. Proc Natl Acad Sci USA 99:2690–2695

    Article  PubMed  CAS  Google Scholar 

  11. 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  PubMed  CAS  Google Scholar 

  12. Newstead S, Kim H, von Heijne G, Iwata S, Drew D (2007) High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 104:13936–13941

    Article  PubMed  CAS  Google Scholar 

  13. Drew D, Newstead S, Sonoda Y, Kim H, von Heijne G, Iwata S (2008) GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat Protoc 3:784–798

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  16. Sonoda Y, Newstead S, Hu NJ, Alguel Y, Nji E, Beis K, Yashiro S, Lee C, Leung J, Cameron AD, Byrne B, Iwata S, Drew D (2011) Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19:17–25

    Article  PubMed  CAS  Google Scholar 

  17. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234

    Article  PubMed  CAS  Google Scholar 

  18. Feilmeier BJ, Iseminger G, Schroeder D, Webber H, Phillips GJ (2000) Green fluorescent protein functions as a reporter for protein localization in Escherichia coli. J Bacteriol 182:4068–4076

    Article  PubMed  CAS  Google Scholar 

  19. Nugent T, Jones DT (2009) Transmembrane protein topology prediction using support vector machines. BMC Bioinformatics 10:159

    Article  PubMed  Google Scholar 

  20. Bernsel A, Viklund H, Hennerdal A, Elofsson A (2009) TOPCONS: consensus prediction of membrane protein topology. Nucleic Acids Res 37:465–468

    Article  Google Scholar 

  21. Luirink J, Yu Z, Wagner S, de Gier JW (2012) Biogenesis of inner membrane proteins in Escherichia coli. Biochim Biophys Acta 1817:965–976

    Article  PubMed  CAS  Google Scholar 

  22. Hsieh JM, Besserer GM, Madej MG, Bui HQ, Kwon S, Abramson J (2010) Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini. Protein Sci 19:868–880

    Article  PubMed  CAS  Google Scholar 

  23. Low C, Jegerschold C, Kovermann M, Moberg P, Nordlund P (2012) Optimisation of over-expression in E. coli and biophysical characterisation of human membrane protein synaptogyrin 1. PLoS One 7:e38244

    Article  PubMed  Google Scholar 

  24. http://www.piercenet.com/browse.cfm?fldID=02020101

  25. Strop P, Brunger AT (2005) Refractive index-based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci 14:2207–2211

    Article  PubMed  CAS  Google Scholar 

  26. Gonzales EB, Kawate T, Gouaux E (2009) Pore architecture and ion sites in acid-sensing ion channels and P2X receptors. Nature 460:599–604

    Article  PubMed  CAS  Google Scholar 

  27. Kawate T, Michel PO, Birdsong WT, Gouaux E (2009) Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature 460:592–598

    Article  PubMed  CAS  Google Scholar 

  28. Guan L, Mirza O, Verner G, Iwata S, Kaback HR (2007) Structural determination of wild-type lactose permease. Proc Natl Acad Sci USA 104:15294–15298

    Article  PubMed  CAS  Google Scholar 

  29. Drew D, Klepsch MM, Newstead S, Flaig R, De Gier JW, Iwata S, Beis K (2008) The structure of the efflux pump AcrB in complex with bile acid. Mol Membr Biol 25:677–682

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Research in the laboratory of JWdG is supported by grants from the Swedish Research Council, the Carl Tryggers Stiftelse, the Marianne and Marcus Wallenberg Foundation, NIH grant 5R01GM081827-03, and the SSF supported Center for Biomembrane Research. David Drew acknowledges the support of the Royal Society (UK) through a University Research Fellowship and the Swedish Research Council.

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Hjelm, A. et al. (2013). Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions. In: Rapaport, D., Herrmann, J. (eds) Membrane Biogenesis. Methods in Molecular Biology, vol 1033. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-487-6_24

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  • DOI: https://doi.org/10.1007/978-1-62703-487-6_24

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-486-9

  • Online ISBN: 978-1-62703-487-6

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