Cell-free Membrane Protein Expression for Solid-State NMR

Methods Mol Biol. 2012;831:85-109. doi: 10.1007/978-1-61779-480-3_6.

Abstract

Although cell-free expression is a relative newcomer to the biochemical toolbox, it has already been reviewed extensively, even in the more specialized cases such as membrane protein expression, nanolipoprotein particles, and applications to crystallography and nuclear magnetic resonance (NMR). Solid-state NMR is also a newcomer to the structural biology toolbox, with its own specificities in terms of sample preparation. Cell-free expression and solid-state NMR are a promising combination that has already proven useful for the structural study of membrane proteins in their native environment, the hydrated lipid bilayer. We describe below several protocols for preparing MscL, a mechanosensitive membrane channel, using cell-free expression destined for a solid-state NMR study. These protocols are flexible and can easily be applied to other membrane proteins, with minor adjustments.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / metabolism
  • Cell-Free System / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / biosynthesis*
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism
  • Ion Channels / biosynthesis*
  • Ion Channels / chemistry
  • Ion Channels / metabolism
  • Isotope Labeling / methods
  • Micelles
  • Nuclear Magnetic Resonance, Biomolecular / methods*

Substances

  • Amino Acids
  • Escherichia coli Proteins
  • Ion Channels
  • Micelles
  • MscL protein, E coli