NMR assignment method for amide signals with cell-free protein synthesis system.
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.
Kohno
Toshiyuki
T
Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan.
D002247:Carbon Isotopes; D002474:Cell-Free System; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012639:Seeds / Q000378:metabolism; D014158:Transcription, Genetic; D014908:Triticum / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
D002247:Carbon Isotopes; D005656:Fungal Proteins; D009587:Nitrogen Isotopes; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011994:Recombinant Proteins; D025801:Ubiquitin