Improving cell-free protein synthesis for stable-isotope labeling |
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Authors: | Takayoshi Matsuda Seizo Koshiba Naoya Tochio Eiko Seki Noriyuki Iwasaki Takashi Yabuki Makoto Inoue Shigeyuki Yokoyama Takanori Kigawa |
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Affiliation: | (1) Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan;(2) Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8502, Japan;(3) Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan |
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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. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. |
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Keywords: | Cell-free protein synthesis In vitro translation Potassium font-variant:small-caps" >d-glutamate Stable-isotope labeling |
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