Improving cell-free protein synthesis for stable-isotope labeling.
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.
Matsuda
Takayoshi
T
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
Koshiba
Seizo
S
Tochio
Naoya
N
Seki
Eiko
E
Iwasaki
Noriyuki
N
Yabuki
Takashi
T
Inoue
Makoto
M
Yokoyama
Shigeyuki
S
Kigawa
Takanori
T
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
D002247:Carbon Isotopes; D005971:Glutamates; D007554:Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D001224:Aspartic Acid; D011392:Proline; D018631:ras Proteins