Wheat Germ Cell-Free Overexpression for the Production of Membrane Proteins

Methods Mol Biol. 2017;1635:91-108. doi: 10.1007/978-1-4939-7151-0_5.

Abstract

Due to their hydrophobic nature, membrane proteins are notoriously difficult to express in classical cell-based protein expression systems. Often toxic, they also undergo degradation in cells or aggregate in inclusion bodies, making delicate issues further solubilization and renaturation. These are major bottlenecks in their structural and functional analysis. The wheat germ cell-free (WGE-CF) system offers an effective alternative not only to classical cell-based protein expression systems but also to other cell-free systems for the expression of membrane proteins. The WGE-CF indeed allows the production of milligram amounts of membrane proteins in a detergent-solubilized, homogenous, and active form. Here, we describe the method to produce a viral integral membrane protein, which is the non-structural protein 2 (NS2) of hepatitis C virus, in view of structural studies by solid-state NMR in a native-like lipid environment.

Keywords: Cell-free protein expression; Detergents; Hepatitis C virus; Protein purification; Wheat germ extract.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System / metabolism*
  • Gene Expression
  • Hepacivirus / metabolism
  • Membrane Proteins / metabolism*
  • Protein Folding
  • Protein Processing, Post-Translational
  • Triticum / metabolism*
  • Viral Nonstructural Proteins / metabolism

Substances

  • Membrane Proteins
  • NS2 protein, Hepatitis C virus
  • Viral Nonstructural Proteins