Chemical biology using the protein-introduced unnatural lysine derivatives as probes

• Project/Area Number: 24550203
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Chemistry related to living body
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Yanagisawa Tatsuo 国立研究開発法人理化学研究所, 横山構造生物学研究室, 研究員 (10450420)
• Project Period (FY): 2012-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2012: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: バイオテクノロジー / 非天然型アミノ酸 / タンパク質 / 翻訳後修飾 / ケミカルバイオロジー / tRNA / ピロリジン / ピロリジルtRNA合成酵素 / ピロリジルtrNA合成酵素 / アミノアシルtRNA合成酵素 / 翻訳

Outline of Final Research Achievements

We developed a novel method to prepare histone proteins bearing multiple N(epsilon)-monomethyllysine residues at specified positions. By the combination of the Release factor 1 (RF1)-knockout (RFzero) Escherichia coli cells and the pair of tRNAPyl and pyrrolysyl-tRNA synthetase (PylRS), a tert-butyloxycarbonyl (Boc)-protected N(epsilon)-monomethyllysine analogue (BocKme1) is translationally incorporated into one or more positions specified with the UAG codon. The Boc groups on the protein are then removed to generate N(epsilon)-monomethyllysine residues. We installed N(epsilon) -monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of histone H3. The present method enables the installation of authentic N(epsilon)-monomethyllysines at multiple positions within a protein for large-scale production and will lead to better understanding of epigenetics research.

Research Products

• [Journal Article] A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys). (2015)
  • Author(s): Yanagisawa, T., Ishii, R., Hikida, Y., Fukunaga, R., Sengoku, T., S. Sekine, Yokoyama,S.
  • Journal Title: J. Struct. Funct. Genomics Volume: 16 Issue: 1 Pages: 25-41
  • DOI: 10.1007/s10969-015-9193-6
  • 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
• [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
• [Presentation] RF1ノックアウト大腸菌のタンパク質発現系および無細胞タンパク質合成系を利用したモノメチルリジンのヒストンへの部位特異的複数導入 (2015)
  • Author(s): 高橋美穂子、柳沢達男、向井崇人、佐藤心、若森昌聡、白水美香子、坂本健作、梅原崇史、横山茂之
  • Organizer: 第10回無細胞生命科学研究会
  • Place of Presentation: 横浜 理研横浜キャンパス
  • Year and Date: 2015-10-13
• [Presentation] 非天然型アミノ酸の部位特異的導入技術とタンパク質科学研究への展開 (2015)
  • Author(s): 柳沢達男、梅原崇史、坂本健作、横山茂之
  • Organizer: 第15回日本蛋白質科学会大会
  • Place of Presentation: 徳島 あわぎんホール
  • Year and Date: 2015-06-24
• [Presentation] 翻訳終結因子RF1欠損大腸菌のタンパク質発現系および無細胞タンパク質合成系を利用したモノメチルリジンのヒストンタンパク質への部位特異的複数導入 (2015)
  • Author(s): ○高橋美穂子、柳沢達男、向井崇人、佐藤心、若森昌聡、白水美香子、坂本健作、梅原崇史、横山茂之
  • Organizer: 日本農芸化学会2015年度大会
  • Place of Presentation: 岡山大学津島キャンパス、岡山
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Expanded genetic code technologies for incorporating lysine residues with naturally-occurring post-translational modifications at multiple sites in proteins. (2015)
  • Author(s): Yanagisawa, T., Fujii, Y., Sakamoto, K., Umehara, T., ○Yokoyama, S.
  • Organizer: RIKEN Epigenetics in Kobe 2015
  • Place of Presentation: 理研CDB、神戸
  • Year and Date: 2015-02-13 – 2015-02-15
• [Presentation] 翻訳終結因子RF1欠損大腸菌のタンパク質発現系および無細胞タンパク質合成系を利用したモノメチルリジンのヒストンタンパク質への部位特異的複数導入 (2014)
  • Author(s): ○柳沢達男、高橋美穂子、向井崇人、佐藤心、若森昌聡、白水美香子、坂本健作、梅原崇史、横山茂之
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 国立京都国際会館、京都
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] Expanded Genetic Code Technologies for Incorporating Modified Lysine at Multiple Sites: 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., Umehara, T., Yokoyama, S.
  • Organizer: 25th tRNA Conference 2014
  • Place of Presentation: Kyllini, Greece
  • Year and Date: 2014-09-21 – 2014-09-25
• [Presentation] Expanded Genetic Code Technologies for Incorporating Modified Lysine at Multiple Sites: 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., Umehara, T., Yokoyama, S.
  • Organizer: The 12th Interdisciplinary Exchange Evening
  • Place of Presentation: 理研和光キャンパス、和光市
  • Year and Date: 2014-09-05
• [Presentation] 翻訳終結因子RF1欠損大腸菌のタンパク質発現系および無細胞タンパク質合成系を利用したモノメチル化リジンのヒストンへの部位特異的複数導入 (2014)
  • Author(s): ○柳沢達男、高橋美穂子、向井崇人、佐藤心、若森昌聡、白水美香子、坂本健作、梅原崇史、横山茂之
  • Organizer: 第16回日本RNA学会年会
  • Place of Presentation: ウインクあいち、名古屋
  • Year and Date: 2014-07-23 – 2014-07-25
• [Presentation] Synthesis of histone proteins with multiple and site-specific monomethyl-lysines (2014)
  • Author(s): ○Umehara, T., Yanagisawa, T., Takahashi, M., Mukai, T., Sato, S., Wakamori, M., Shirouzu, M., Sakamoto, K., Yokoyama, S.
  • Organizer: FASEB meeting (2014): Biological Methylation: Regulation of Chromatin, Epigenetics, and Disease
  • Place of Presentation: Nassau, Bahamas
  • Year and Date: 2014-07-06 – 2014-07-11
• [Presentation] 翻訳終結因子欠損大腸菌のタンパク質発現系および無細胞タンパク質合成系を利用したモノメチル化リジンのヒストンへの部位特異的複数導入 (2014)
  • Author(s): ○柳沢達男、高橋美穂子、向井崇人、佐藤心、若森昌聡、白水美香子、坂本健作、梅原崇史、横山茂之
  • Organizer: 第9回ケミカルバイオロジー学会大会
  • Place of Presentation: 大阪大学豊中キャンパス、大阪
  • Year and Date: 2014-06-11 – 2014-06-13
• [Presentation] In vivo and in vitro site-specific incorporation/installation of non-canonical lysine derivatives into proteins by using pyrrolysyl-tRNA synthetase-tRNAPyl pair and its application for epigenetic studies (2014)
  • Author(s): ○Tatsuo Yanagisawa, Mihoko Takahashi, Shin Sato, Takahito Mukai, Masatoshi Wakamori, Mikako Shirouzu, Takashi Umehara, Kensaku Sakamoto, and Shigeyuki Yokoyama
  • Organizer: RIKEN EPIGENETICS in Yokohama
  • Place of Presentation: 横浜、理研横浜キャンパス
• [Presentation] Crystallographic studies on specific aminoacylation of tRNA and genetic code expansion (2013)
  • Author(s): Naganuma M, Sasaki HM, Mukai T, Yanagisawa T, Ohtake K, Wakamori M, Hikida Y, Higashijima K, Shibata R, Nishimoto M, Bessho Y, Chong YE, Guo M, Yang XL, Shimmel P, Fukunaga R, Harada Y, Kimoto M, Adachi J, Hino N, Sato A, Hayashi A, Terada T, Umehara T, Ito T, Sekine S, Hirao I, Shirouzu M, Sakamoto K, ○Yokoyama S.
  • Organizer: 7th International Conference on Structural Genomics 2013
  • Place of Presentation: 札幌、京王プラザホテル
• [Presentation] Synthesis of histone with multiple site-specific monomethylation for reconstitution of epigenetic nucleosome (2013)
  • Author(s): ○Mihoko Takahashi, Tatsuo Yanagisawa, Shin Sato, Masatoshi Wakamori, Takahito Mukai, Mikako Shirouzu, Kensaku Sakamoto1, Shigeyuki Yokoyama, Takashi Umehara
  • Organizer: The 65th Fujihara Seminar; International Symposium on Synthetic Biology of Unnatural Base Pairs and Amino Acids
  • Place of Presentation: 苫小牧、グランドホテルニュー王子