Skip to main content
SpringerLink
Log in

Cell-Free Membrane Protein Expression for Solid-State NMR

  • Protocol
  • First Online:
Protein NMR Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 831))

Abstract

Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments.

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. Lyford, L. K., and Rosenberg, R. L. (1999) Cell-free expression and functional reconstitution of Homo-oligomeric α7 Nicotinic Acetylcholine Receptors into Planar Lipid Bilayers. J. Biol. Chem. 274, 25675–25681.

    Article  PubMed  CAS  Google Scholar 

  2. Elbaz, Y., Steiner-Mordoch, S., Danieli, T., and 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, 1519–1524.

    Article  PubMed  CAS  Google Scholar 

  3. Berrier, C., Park, K. H., Abes, S., Bibonne, A., Betton, J. M., and 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, 12585–12591.

    Article  PubMed  CAS  Google Scholar 

  4. Klammt, C., Lohr, F., Schafer, B., Haase, W., Dötsch, V., Ruterjans, H., Glaubitz, C., and Bernhard, F. (2004) High level cell-free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. 271, 568–580.

    Article  PubMed  CAS  Google Scholar 

  5. Wagner, S., Bader, M. L., Drew, D., and de Gier, J. W. (2006) Rationalizing membrane protein overexpression. Trends Biotechnol. 24, 364–371.

    Article  PubMed  CAS  Google Scholar 

  6. Eshaghi, S. (2009) High-throughput expression and detergent screening of integral membrane proteins. Methods Mol. Biol. 498, 265–271.

    Article  PubMed  CAS  Google Scholar 

  7. Tate, C. G. (2010) Practical considerations of membrane protein instability during purification and crystallisation. Methods Mol. Biol. 601, 187–203.

    Article  PubMed  CAS  Google Scholar 

  8. Breyton, C., Pucci, B., and Popot, J.-L. (2010) Amphipols and fluorinated surfactants: Two alternatives to detergents for studying membrane proteins in vitro. Methods Mol. Biol. 601, 219–245.

    Article  PubMed  CAS  Google Scholar 

  9. White, S. H. (2010) Membrane Proteins of Known Structure. University of California at Irvine. http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html Accessed 25 July 2011.

  10. Warschawski, D. E. (2010) Membrane Proteins of Known Structure Determined by NMR. Drorlist. http://www.drorlist.com/nmr/MPNMR.html Accessed 25 July 2011.

  11. Schneider, B., Junge, F., Shirokov, V. A., Durst, F., Schwarz, D., Dötsch, V., and Bernhard, F. (2010) Membrane protein expression in cell-free systems. Methods Mol. Biol. 601, 165–186.

    Article  PubMed  CAS  Google Scholar 

  12. Sobhanifar, S., Reckel, S., Junge, F., Schwarz, D., Kai, L., Karbyshev, M., Löhr, F., Bernhard, F., and Dötsch, V. (2010) Cell-free expression and stable isotope labelling strategies for membrane proteins. J. Biomol. NMR 46, 33–43.

    Article  PubMed  CAS  Google Scholar 

  13. Lehner, I., Basting, D., Meyer, B., Haase, W., Manolikas, T., Kaiser, C., Karas, M., and Glaubitz, C. (2008) The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric. J. Biol. Chem. 283, 3281–3288.

    Article  PubMed  CAS  Google Scholar 

  14. Abdine, A., Verhoeven, M. A., Park, K.-H., Ghazi, A., Guittet, E., Berrier, C., Van Heijenoort, C., and Warschawski, D. E. (2010) Structural study of the membrane protein MscL using cell-free expression and solid-state NMR. J. Magn. Reson. 204, 155–159.

    Article  PubMed  CAS  Google Scholar 

  15. Kalmbach, R., Chizhov, I., Schumacher, M. C., Friedrich, T., Bamberg, E., and Engelhard, M. (2007) Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J. Mol. Biol. 371, 639–648.

    Article  PubMed  CAS  Google Scholar 

  16. Marques, B., Liguori, L., Paclet, M. H., Villegas-Mendéz, A., Rothe, R., Morel, F., Lenormand, J.-L. (2007) Liposome-mediated cellular delivery of active gp91(phox). PLoS One 2, e856.

    Article  PubMed  Google Scholar 

  17. Katzen, F., Fletcher, J. E., Yang, J. P., Kang, D., Peterson, T. C., Cappuccio, J. A., Blanchette, C. D., Sulchek, T., Chromy, B. A., Hoeprich, P. D., Coleman, M. A., and Kudlicki, W. (2008) Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach. J. Proteome Res. 7, 3535–3542.

    Article  PubMed  CAS  Google Scholar 

  18. Berrier, C., Guilvout, I., Bayan, N., Park, K.-H., Mesneau, A., Chami, M., Pugsley, A. P., and Ghazi, A. (2011) Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL - MscL does not need the insertase YidC for insertion in vitro. Biochim. Biophys. Acta 1808, 41–46.

    Article  PubMed  CAS  Google Scholar 

  19. Abdine, A., Verhoeven, M. A., and Warschawski, D. E. (2011) Cell-free expression and labeling strategies for a new decade in solid-state NMR. New Biotechnol. 28, 272–276.

    Article  CAS  Google Scholar 

  20. He, M. (2008) Cell-free protein synthesis: applications in proteomics and biotechnology. New Biotechnol. 25, 126–132.

    Article  CAS  Google Scholar 

  21. Rigaud, J.-L., and Lévy, D. (2003) Reconstitution of Membrane Proteins into Liposomes. Methods Enzymol. 372, 65–86.

    Article  PubMed  CAS  Google Scholar 

  22. Triba, M. N., Zoonens, M., Popot, J.-L., Devaux, P. F., and Warschawski, D. E. (2006) Reconstitution and alignment by a magnetic field of a β-barrel membrane protein in bicelles. Eur. Biophys. J. 35, 268–275.

    Article  PubMed  CAS  Google Scholar 

  23. Leitz, A. J., Bayburt, T. H., Barnakov, A. N., Springer, B. A., and Sligar, S. G. (2006) Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology. BioTechniques 40, 601–612.

    Article  PubMed  CAS  Google Scholar 

  24. De Grip, W. J., Van Oostrum, J., and Bovee-Geurts, P. H. M. (1998) Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes. Biochem. J. 330, 667–674.

    Google Scholar 

  25. Sukharev, S. I., Schroeder, M. J., and McCaslin, D. R. (1999) Stoichiometry of the Large Conductance Bacterial Mechanosensitive Channel of E. coli. A Biochemical Study. J. Membrane Biol. 171, 183–193.

    Article  CAS  Google Scholar 

  26. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.

    Article  PubMed  CAS  Google Scholar 

  27. Wiechelman, K. J., Braun, R. D., and Fitzpatrick, J. D. (1988) Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal. Biochem. 175, 231–237.

    Article  PubMed  CAS  Google Scholar 

  28. Rouser, G., Fkeischer, S., and Yamamoto, A. (1970) Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494–496.

    Article  PubMed  CAS  Google Scholar 

  29. Laird, D. M., Eble, K. S., and Cunningham, C. C. (1986) Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside. J. Biol. Chem. 261, 14844–14850.

    PubMed  CAS  Google Scholar 

  30. Etezady-Esfarjani, T., Hiller, S., Villalba, C., and Wüthrich, K. (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J. Biomol. NMR 39, 229–238.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche and the Fondation pour la Recherche Médicale (to A.A.), by the CNRS (UMR 7099 and 8619), the ANR (ANR-06-JCJC0014), the Univ Paris Diderot and the Université Paris-Sud 11. We thank Alexandre Ghazi, Catherine Berrier, Emmanuelle Billon-Denis, and Michiel A. Verhoeven for helping us optimize the protocols presented here.

Author information

Authors and Affiliations

  1. CNRS and Université Paris Diderot, IBPC, Paris, France

    Alaa Abdine, Kyu-Ho Park & Dror E. Warschawski

Authors
  1. Alaa Abdine
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kyu-Ho Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dror E. Warschawski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dror E. Warschawski .

Editor information

Editors and Affiliations

  1. University of Albany, State University of New York, Washington Avenue 1400, Albany, 12222, New York, USA

    Alexander Shekhtman

  2. University of Albany, State University of New York, Washington Avenue 1400, Albany, 12222, New York, USA

    David S. Burz

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Abdine, A., Park, KH., Warschawski, D.E. (2012). Cell-Free Membrane Protein Expression for Solid-State NMR. In: Shekhtman, A., Burz, D. (eds) Protein NMR Techniques. Methods in Molecular Biology, vol 831. Humana Press. https://doi.org/10.1007/978-1-61779-480-3_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-480-3_6

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-479-7

  • Online ISBN: 978-1-61779-480-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation