Advertisement

Cell-Free Production of Membrane Proteins in Escherichia coli Lysates for Functional and Structural Studies

  • Ralf-Bernhardt Rues
  • Erik Henrich
  • Coilin Boland
  • Martin Caffrey
  • Frank BernhardEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1432)

Abstract

The complexity of membrane protein synthesis is largely reduced in cell-free systems and it results into high success rates of target expression. Protocols for the preparation of bacterial lysates have been optimized in order to ensure reliable efficiencies in membrane protein production that are even sufficient for structural applications. The open accessibility of the semisynthetic cell-free expression reactions allows to adjust membrane protein solubilization conditions according to the optimal folding requirements of individual targets. Two basic strategies will be exemplified. The post-translational solubilization of membrane proteins in detergent micelles is most straightforward for crystallization approaches. The co-translational integration of membrane proteins into preformed nanodiscs will enable their functional characterization in a variety of natural lipid environments.

Key words

G-protein-coupled receptors Nanodiscs Synthetic biology Membranes Membrane protein crystallization Lipid screening 

Notes

Acknowledgments

This work was funded by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG) and by the German Ministry of Education and Science (BMBF). Support was further obtained by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI).

References

  1. 1.
    Henrich E, Hein C, Dötsch V et al (2015) Membrane protein production in Escherichia coli cell-free lysates. FEBS Lett 589:1713–1722CrossRefPubMedGoogle Scholar
  2. 2.
    Hein C, Henrich E, Orbán E et al (2014) Hydrophobic supplements in cell‐free systems: designing artificial environments for membrane proteins. Eng Life Sci 14:365–379CrossRefGoogle Scholar
  3. 3.
    Junge F, Haberstock S, Roos C et al (2011) Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins. N Biotechnol 28:262–271CrossRefPubMedGoogle Scholar
  4. 4.
    Kigawa T, Yabuki T, Yoshida Y et al (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett 442:15–19CrossRefPubMedGoogle Scholar
  5. 5.
    Boland C, Li D, Shah ST et al (2014) Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination. Cell Mol Life Sci 71:4895–4910CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Proverbio D, Roos C, Beyermann M et al (2013) Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments. Biochim Biophys Acta 1828:2182–2192CrossRefPubMedGoogle Scholar
  7. 7.
    Li Y, Wang E, Wang Y (1999) A modified procedure for fast purification of T7 RNA polymerase. Protein Expr Purif 16:355–358CrossRefPubMedGoogle Scholar
  8. 8.
    Denisov IG, Grinkova YV, Lazarides AA et al (2004) Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J Am Chem Soc 126:3477–3487CrossRefPubMedGoogle Scholar
  9. 9.
    Berrier C, Guilvout I, Bayan N et al (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 1808:41–46Google Scholar
  10. 10.
    Haberstock S, Roos C, Hoevels Y et al (2012) A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems. Protein Expr Purif 82:308–316CrossRefPubMedGoogle Scholar
  11. 11.
    Schneider B, Junge F, Shirokov VA et al (2010) Membrane protein expression in cell-free systems. Methods Mol Biol 601:165–186CrossRefPubMedGoogle Scholar
  12. 12.
    Soubias O, Gawrisch K (2012) The role of the lipid matrix for structure and function of the GPCR rhodopsin. Biochim Biophys Acta 1818:234–240CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ralf-Bernhardt Rues
    • 1
  • Erik Henrich
    • 1
  • Coilin Boland
    • 2
  • Martin Caffrey
    • 2
  • Frank Bernhard
    • 1
    Email author
  1. 1.Centre for Biomolecular Magnetic Resonance, Institute for Biophysical ChemistryGoethe-University of Frankfurt/MainFrankfurt/MainGermany
  2. 2.Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and ImmunologyTrinity College DublinDublinIreland

Personalised recommendations