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  3. Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins

  • Protein Expr Purif. 2015 Jan;105:39-46. doi: 10.1016/j.pep.2014.10.003.
Marie-Laure Fogeron 1,  Aurélie Badillo 2,  Vlastimil Jirasko 3,  Jérôme Gouttenoire 4,  David Paul 5,  Loick Lancien 1,  Darius Moradpour 4,  Ralf Bartenschlager 3,  Beat H Meier 6,  François Penin 7,  Anja Böckmann 8
Affiliations

Affiliations

  • 1 Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367 Lyon, France.
  • 2 Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367 Lyon, France; RD-Biotech, Recombinant Protein Unit, 3 rue Henri Baigue, 25000 Besançon, France.
  • 3 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany; German Centre for Infection Research (DZIF), Partner Site Heidelberg, Heidelberg, Germany.
  • 4 Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, 1011 Lausanne, Switzerland.
  • 5 Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
  • 6 Physical Chemistry, ETH Zurich, 8093 Zurich, Switzerland.
  • 7 Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367 Lyon, France. Electronic address: [email protected].
  • 8 Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367 Lyon, France. Electronic address: [email protected].
PMID: 25306874 DOI: 10.1016/j.pep.2014.10.003
Abstract

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Keywords

Cell-free protein expression; Detergents; Hepatitis C virus proteins.

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