Novel protein-synthesis technology achieving the installation of posttranslational modifications and other chemical modifications through genetic code expansion

• Project/Area Number: 26291035
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Biophysics
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: SAKAMOTO KENSAKU 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, グループディレクター (50240685)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥13,390,000 (Direct Cost: ¥10,300,000、Indirect Cost: ¥3,090,000); Fiscal Year 2017: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2016: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2015: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2014: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
• Keywords: 拡張遺伝暗号 / 非天然型アミノ酸 / アミノアシルtRNA合成酵素 / 合成生物学 / カイコ / シルク / アジドフェニルアラニン / 蛋白質 / 遺伝暗号 / タンパク質工学 / タンパク質生産 / 翻訳後修飾 / 翻訳終結因子 / コドン再定義 / ホモアルギニン / ヌクレオソーム / RF-1 / ニトロチロシン / UAGコドン

Outline of Final Research Achievements

We developed a novel protein-synthesis technology that allows the incorporation of posttranslational modifications and other chemical modifications into protein at desired sites. It was one of the primary target of this study to collaborate with scientists in other fields and demonstrate the usefulness of the developed technology. To increase the productivity of proteins containing non-natural amino acids, release factor I was eliminated from E. coli host cells, accompanied by a large-scale genomic modification, and this engineering greatly facilitated the incorporation of nitrotyrosine, acetyllysine and other posttranslational modifications. Through the collaboration with NARO, an agriculture-related institute, the genetic code of silkworms was modified in a way that led to a great increase of the yield of silk with azido groups, useful for industrial purposes.

Research Products

• [Journal Article] Genetic Code Expansion of the Silkworm Bombyx mori to Functionalize Silk Fiber (2018)
  • Author(s): Teramoto H, Amano Y, Iraha F, Kojima K, Ito T, Sakamoto K
  • Journal Title: ACS Synthetic Biology Volume: 7 Issue: 3 Pages: 801-806
  • DOI: 10.1021/acssynbio.7b00437
  • Peer Reviewed
• [Journal Article] Adenovirus vector-based incorporation of a photo-cross-linkable amino acid into proteins in human primary cells and cancerous cell lines. (2016)
  • Author(s): Kita A, Hino N, Higashi S, Hirota K, Narumi R, Adachi J, Takafuji K, Ishimoto K, Okada Y, Sakamoto K, Tomonaga T, Takashima S, Mizuguchi H, Doi T.
  • Journal Title: Sci Rep Volume: 6 Issue: 1 Pages: 36946-36946
  • DOI: 10.1038/srep36946
  • Peer Reviewed / Open Access
• [Journal Article] 大腸菌の遺伝暗号の改変がもたらす組換えタンパク質生産技術の革新 (2016)
  • Author(s): 坂本健作
  • Journal Title: 化学と生物（日本農芸化学会） Volume: 54(5) Pages: 343-350
  • NAID: 130006772546
  • Peer Reviewed
• [Journal Article] Crystal structure of eukaryotic translation initiation factor 2B (2016)
  • Author(s): Kazuhiro Kashiwagi, Mari Takahashi, Madoka Nishimoto, Takuya B. Hiyama, Toshiaki Higo, Takashi Umehara, Kensaku Sakamoto, Takuhiro Ito, Shigeyuki Yokoyama
  • Journal Title: Nature Volume: 531 Issue: 7592 Pages: 122-125
  • DOI: 10.1038/nature16991
  • Peer Reviewed
• [Journal Article] Incorporation of a Doubly Functionalized Synthetic Amino Acid into Proteins for Creating Chemical and Light-Induced Conjugates (2016)
  • Author(s): Atsushi Yamaguchi, Takayoshi Matsuda, Kazumasa Ohtake, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Yoshihisa Fujiwara, Takayoshi Watanabe, Takahiro Hohsaka, Kensaku Sakamoto
  • Journal Title: Bioconjugate Chemistry Volume: 27 Issue: 1 Pages: 198-206
  • DOI: 10.1021/acs.bioconjchem.5b00602
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] 非天然型アミノ酸アミノ酸の導入によるタンパク質の安定化技術 (2016)
  • Author(s): 大竹和正、坂本健作
  • Journal Title: バイオサイエンスとインダストリー Volume: 74 Pages: 34-36
• [Journal Article] Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation (2015)
  • Author(s): Masatoshi Wakamori, Yoshifumi Fujii, Noriyuki Suka, Mikako Shirouzu, Kensaku Sakamoto, Takashi Umehara, Shigeyuki Yokoyama
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 17204-17204
  • DOI: 10.1038/srep17204
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli (2015)
  • Author(s): Mukai, T., Yamaguchi, A., Ohtake, K., Takahashi, M., Hayashi, A., Iraha, F., Kira, S., Yanagisawa, T., Yokoyama, S., Hoshi, H., Kobayashi, T., Sakamoto, K.
  • Journal Title: Nucleic Acids Research Volume: 43(16) Issue: 16 Pages: 8111-8122
  • DOI: 10.1093/nar/gkv787
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Protein stabilization utilizing a redefined codon (2015)
  • Author(s): Ohtake, K., Yamaguchi, A., Mukai, T., Kashimura, H., Hirano, N., Haruki, M., Kohashi, S., Yamagishi, K., Murayama. K., Tomabechi, Y., Itagaki, T., Akasaka, R., Kawazoe, M., Takemoto, C., Shirouzu, M., Yokoyama, S. & Sakamoto, K.
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 9762-9762
  • DOI: 10.1038/srep09762
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Highly reproductive Escherichia coli cells with a blank assignment for the UAG codon (2015)
  • Author(s): Mukai, T., Hoshi, H.; Ohtake, K., Takahashi, M., Yamaguchi, A., Hayashi, A., Yokoyama, S. & Sakamoto, K.
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 9699-9699
  • DOI: 10.1038/srep09699
  • Peer Reviewed / Open Access
• [Journal Article] Ubiquitin acetylation inhibits polyubiquitin chain elongation. (2015)
  • Author(s): Ohtake F, Saeki Y, Sakamoto K, Ohtake K, Nishikawa H, Tsuchiya H, Ohta T, Tanaka K, Kanno J.
  • Journal Title: EMBO Rep. Volume: 16 Issue: 2 Pages: 192-201
  • DOI: 10.15252/embr.201439152
  • Peer Reviewed / Open Access
• [Journal Article] Expanded genetic code technologies for modified lysine installation at multiple sites (2014)
  • Author(s): Yanagisawa, T., Umehara, T., Sakamoto, K., and Yokoyama, S.
  • Journal Title: ChemBioChem Volume: 15 Issue: 15 Pages: 2181-2187
  • DOI: 10.1002/cbic.201402266
  • Peer Reviewed
• [Journal Article] Structural Stability of Halogenated Protein: Fragment Molecular Orbital Study (2014)
  • Author(s): Jumpei Takanohashi, Sousuke Kohashi, Takeshi Ishikawa, Kensaku Sakamoto, Kenji Yamagishi
  • Journal Title: J. Comput. Chem. Jpn Volume: 13 Pages: 308-309
  • NAID: 130004933669
  • Peer Reviewed
• [Journal Article] Multiple site-specific installations of N(ε)-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis (2014)
  • Author(s): Yanagisawa, T., Takahashi, M., Mukai, T., Sato, S., Wakamori, M., Shirouzu, M., Sakamoto, K., and Umehara, T. and Yokoyama, S.
  • Journal Title: ChemBioChem Volume: 15 Issue: 12 Pages: 1830-1838
  • DOI: 10.1002/cbic.201402291
  • Peer Reviewed / Acknowledgement Compliant
• [Presentation] Expanded genetic codes in mammalian cells, codon reassignments in E. coli, and their applications (2016)
  • Author(s): Kensaku Sakamoto
  • Organizer: The 2016 Genetic Code Expansion Conference
  • Place of Presentation: アメリカ コバリス
  • Year and Date: 2016-08-11
  • Int'l Joint Research / Invited