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An unnatural base pair for incorporating amino acid analogs into proteins

Nature Biotechnology volume 20, pages 177182 (2002) | Download Citation

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Abstract

An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography–mass spectrometry (LC-MS) analysis of the products. This coupled transcription–translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.

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Acknowledgements

We thank T. Masaki, of Ibaraki University, for providing the Achromobacter Protease I, and R. Ishitani, The University of Tokyo, for helpful discussion.

Author information

Author notes

    • Ichiro Hirao
    • , Tsuneo Mitsui
    •  & Shigeyuki Yokoyama

    Current address: Protein Preparation/NMR Facilities, Genomic Sciences Center, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

Affiliations

  1. Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

    • Ichiro Hirao
    • , Tsuyoshi Fujiwara
    • , Tsuneo Mitsui
    • , Tomoko Yokogawa
    • , Taeko Okuni
    •  & Shigeyuki Yokoyama
  2. Genomic Sciences Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.

    • Takashi Ohtsuki
    • , Takashi Yabuki
    • , Takanori Kigawa
    • , Koichiro Kodama
    •  & Shigeyuki Yokoyama
  3. Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.

    • Hiroshi Nakayama
    •  & Koji Takio
  4. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

    • Koichiro Kodama
    •  & Shigeyuki Yokoyama
  5. Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagito, Gifu 501-1193, Japan.

    • Takashi Yokogawa
    •  & Kazuya Nishikawa

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Correspondence to Ichiro Hirao or Shigeyuki Yokoyama.

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DOI

https://doi.org/10.1038/nbt0202-177

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