title: Improving cell-free protein synthesis for stable-isotope labeling.
abstract: Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium L-glutamate (L-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium D-glutamate (D-Glu system). The productivity of the D-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H-15N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the D-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the D-Glu system. These results indicate that the D-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins.
authors.affiliation: Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Seizo
authors.initials: S
authors.lastname: Koshiba
authors.forename: Naoya
authors.initials: N
authors.lastname: Tochio
authors.forename: Eiko
authors.initials: E
authors.lastname: Seki
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Iwasaki
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Makoto
authors.initials: M
authors.lastname: Inoue
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry*; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D018631:ras Proteins / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D005971:Glutamates; D007554:Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D001224:Aspartic Acid; D011392:Proline; D018631:ras Proteins