NMR assignment method for amide signals with cell-free protein synthesis system

Methods Mol Biol. 2010;607:113-26. doi: 10.1007/978-1-60327-331-2_11.

Abstract

Nuclear magnetic resonance (NMR) methods are widely used to determine the three-dimensional structures of proteins, to estimate protein folding, and to discover high-affinity ligands for proteins. However, one of the problems to apply such NMR methods to proteins is that we should obtain mg quantities of (15)N and/or (13)C labeled pure proteins of interest. Here, we describe the method to produce dual amino acid-selective (13)C-(15)N labeled proteins for NMR study using the improved wheat germ cell-free system, which enables sequence-specific assignments of amide signals simply even for very large protein.

MeSH terms

  • Carbon Isotopes
  • Cell-Free System
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / chemistry
  • Fungal Proteins / genetics
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular*
  • Protein Biosynthesis
  • Protein Conformation
  • Protein Engineering / methods*
  • Protein Processing, Post-Translational*
  • Protein Sorting Signals*
  • RNA, Messenger / biosynthesis
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Seeds / metabolism
  • Transcription, Genetic
  • Triticum / embryology
  • Triticum / genetics
  • Triticum / metabolism*
  • Ubiquitin / metabolism*
  • Ubiquitination

Substances

  • Carbon Isotopes
  • Fungal Proteins
  • Nitrogen Isotopes
  • Protein Sorting Signals
  • RNA, Messenger
  • Recombinant Proteins
  • Ubiquitin