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High-Level Cell-Free Production of Membrane Proteins with Nanodiscs

  • Christian Roos
  • Lei Kai
  • Stefan Haberstock
  • Davide Proverbio
  • Umesh Ghoshdastider
  • Yi Ma
  • Slawomir Filipek
  • Xiaoning Wang
  • Volker Dötsch
  • Frank Bernhard
Part of the Methods in Molecular Biology book series (MIMB, volume 1118)

Abstract

This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.

Key words

Nanodiscs Tag variation screen Membrane proteins Expression optimization Lipid screen Reconstitution 

Notes

Acknowledgements

This work was supported by the Collaborative Research Center (SFB) 807 of the German Research Foundation (DFG). We further thank the European Drug Initiative on Channels and Transporters (EDICT), contract number HEALTH-F4-2007-201924; the European initiative on Structural Biology of Membrane Proteins (SBMP), contract number PITN-GA-2008-211800; and the NIH (grant number U54 GM094608) for funding. We further thank Oliver Ernst for helpful advice and Vladimir Shirokov for critical discussions.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Christian Roos
    • 1
  • Lei Kai
    • 1
  • Stefan Haberstock
    • 1
  • Davide Proverbio
    • 1
  • Umesh Ghoshdastider
    • 1
    • 2
  • Yi Ma
    • 1
    • 3
  • Slawomir Filipek
    • 2
  • Xiaoning Wang
    • 3
  • Volker Dötsch
    • 1
  • Frank Bernhard
    • 1
  1. 1.Centre for Biomolecular Magnetic Resonance, Institute for Biophysical ChemistryGoethe-University of Frankfurt/MainFrankfurt/MainGermany
  2. 2.Faculty of ChemistryUniversity of WarsawWarsawPoland
  3. 3.School of Bioscience and BioengineeringSouth China University of TechnologyGuangzhouPeople’s Republic of China

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