Cell-Free Protein Production pp 187-212

Part of the Methods in Molecular Biology book series (MIMB, volume 607)

Strategies for the Cell-Free Expression of Membrane Proteins

  • Sina Reckel
  • Solmaz Sobhanifar
  • Florian Durst
  • Frank Löhr
  • Vladimir A. Shirokov
  • Volker Dötsch
  • Frank Bernhard
Protocol

Abstract

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.

Key words:

Continuous exchange cell-free expression S30 extract P-CF D-CF L-CF Detergents Lipids NMR Transmembrane segments Stable isotope labelling 

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Copyright information

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

Authors and Affiliations

  • Sina Reckel
    • 1
  • Solmaz Sobhanifar
    • 1
  • Florian Durst
    • 1
  • Frank Löhr
    • 1
  • Vladimir A. Shirokov
    • 2
  • Volker Dötsch
    • 1
  • Frank Bernhard
    • 1
  1. 1.Centre for Biomolecular Magnetic ResonanceInstitute for Biophysical Chemistry, University of Frankfurt/MainFrankfurt/MainGermany
  2. 2.Institute of Protein Research, Russian Academy of SciencesPushchinoRussia

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