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An unnatural base pair for incorporating amino acid analogs into proteins

Nature Biotechnology volume 20, pages 177–182 (2002)Cite this article

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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Figure 1
Figure 2: T7 transcription employing s·y pairing.
Figure 3: The transcription–translation system involving the s·y base pair.
Figure 4: Preparation of aminoacylated tRNACUs.
Figure 5: Incorporation of ClTyr into the Ras protein.

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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.

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Author notes

  1. Ichiro Hirao, Tsuneo Mitsui & Shigeyuki Yokoyama

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

Authors and Affiliations

  1. Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa, Wako-shi, 351-0198, Saitama, 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, 230-0045, Kanagawa, Japan

    Takashi Ohtsuki, Takashi Yabuki, Takanori Kigawa, Koichiro Kodama & Shigeyuki Yokoyama

  3. Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, 351-0198, Saitama, 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, 113-0033, Tokyo, 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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Hirao, I., Ohtsuki, T., Fujiwara, T. et al. An unnatural base pair for incorporating amino acid analogs into proteins. Nat Biotechnol 20, 177–182 (2002). https://doi.org/10.1038/nbt0202-177

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