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. Epub 2014 Oct 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System*
  • Detergents / chemistry*
  • Hepacivirus / genetics*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Micelles
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*
  • Triticum / metabolism*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • Detergents
  • Membrane Proteins
  • Micelles
  • Recombinant Proteins
  • Viral Proteins