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Cell-Free Protein Production

Volume 607 of the series Methods in Molecular Biology pp 187-212

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Strategies for the Cell-Free Expression of Membrane Proteins

  • Sina ReckelAffiliated withCentre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main
  • , Solmaz SobhanifarAffiliated withCentre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main
  • , Florian DurstAffiliated withCentre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main
  • , Frank LöhrAffiliated withCentre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main
  • , Vladimir A. ShirokovAffiliated withInstitute of Protein Research, Russian Academy of Sciences
  • , Volker DötschAffiliated withCentre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main
  • , Frank BernhardAffiliated withCentre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt/Main Email author 

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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