Site-specific incorporation of non-natural amino acids into proteins by using pyrrolysyl-tRNA synthetase

• Project/Area Number: 21550167
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Chemistry related to living body
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: YANAGISAWA Tatsuo 独立行政法人理化学研究所, システム研究チーム, 研究員 (10450420)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2011: ¥130,000 (Direct Cost: ¥100,000、Indirect Cost: ¥30,000); Fiscal Year 2010: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2009: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
• Keywords: バイオテクノロジー / 非天然型アミノ酸 / タンパク質 / クロスリンカー / ケミカルバイオロジー / 翻訳 / ピロリジン / アミノアシルtRNA合成酵素 / tRNA / 翻訳後修飾 / アミノアシル-tRNA合成酵素 / 有用性官能基 / ピロリジル-tRNA合成酵素

Research Abstract

Site-specific incorporation of N^ε-(p-trifluoreomethyldiazirinylbenzyloxycarbonyl) lysine(TmdZLys), a new lysine derivative with a diazirine photo-crosslinker, into proteins has been achieved by the pyrrolysyl-tRNA synthetases(PylRS)/tRNA^<Pyl> system. The incorporated TmdZLys enabled wide-range photo-crosslinking with target proteins. This system is useful for probing protein interaction networks in living cells.

Research Products

• [Journal Article] (2012)
  • Author(s): Yanagisawa, T., Hino, N., Iraha, F., Mukai, T., Sakamoto, K., Yokoyama, S.
  • Journal Title: Mol. BioSyst Volume: 8 Pages: 1131-1135
  • Peer Reviewed
• [Journal Article] Wade-range protein photo-crosslinking achieved by a genetically encoded N^ε-(benzyloxycarbonyl) lysine derivative with a diazirinyl moiety (2012)
  • Author(s): T. Yanagisawa, N. Hino, F. Iraha, T. Mukai, K. Sakamoto, S. Yokoyama
  • Journal Title: Mol. Bio Syst. Volume: 8 Issue: 4 Pages: 1131-1135
  • DOI: 10.1039/c2mb05321g
  • Peer Reviewed
• [Journal Article] (2011)
  • Author(s): Mukai, T., Yanagisawa, T., Ohtake, K., Wakamori, M., Adachi, J., Hino, N., Sato, A., Kobayashi, T., Hayashi, A., Shirouzu, M., Umehara, T., Yokoyama, S., Sakamoto, K.
  • Journal Title: Biochem. Biophys. Res. Commun Volume: 411 Pages: 757-761
  • Peer Reviewed
• [Journal Article] Genetic-code evolution for protein synthesis with non-natural amino acids (2011)
  • Author(s): T. Mukai, T. Yanagisawa, K. Ohtake, M. Wakamori, J. Adachi, N. Hino, A. Sato, T. Kobayashi, A. Hayashi, M. Shirouzu, T. Umehara, S. Yokoyama, K. Sakamoto
  • Journal Title: Biochem. Biophys. Res. Comm. Volume: 411 Pages: 757-761
  • Peer Reviewed
• [Journal Article] A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P (2010)
  • Author(s): T. Yanagisawa. T. Sumida, R. Ishii, C. Takemoto, S. Yokoyama
  • Journal Title: Nat. Struct. Mol. Biol. Volume: 17 Pages: 1136-1143
  • Peer Reviewed
• [Journal Article] Crystallizaton and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P (2010)
  • Author(s): T. Sumida, T. Yanagisawa, R. Ishii, S. Yokoyama
  • Journal Title: Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commn. Volume: 66 Pages: 1115-1119
  • Peer Reviewed
• [Presentation] 終止コドンUAGがセンス・コドンに再定義された大腸菌株を用いた非天然型アミノ酸のタンパク質への導入 (2011)
  • Author(s): 坂本健作、安達仁朗、若森昌聡、白水美香子、梅原崇史、柳沢達男、向井崇人、横山茂之
  • Organizer: 第6回無細胞生命科学研究会
  • Place of Presentation: 兵庫
  • Year and Date: 2011-11-16
• [Presentation] 転移RNAを擬態する大腸菌翻訳因子EF-Pのアミノアシル化修飾 (2011)
  • Author(s): 柳沢達男、澄田智美、石井亮平、竹本千重、横山茂之
  • Organizer: 第5回細菌学若手コロッセウム
  • Place of Presentation: 高知県高知市
• [Presentation] 終止コドンUAGがセンス・コドンに再定義された大腸菌株を用いた非天然型アミノ酸のタンパク質への導入 (2011)
  • Author(s): 坂本健作、安達仁朗、若森昌聡、白水美香子、梅原崇史、柳沢達男、向井崇人、横山茂之
  • Organizer: 第6回無細胞生命科学研究会
  • Place of Presentation: 兵庫県姫路市
• [Presentation] 大腸菌、昆虫細胞、動物細胞を用いた非天然型アミノ酸のタンパク質への部位特異的導入法の開発 (2010)
  • Author(s): 坂本健作、向井崇人、大竹和正、伊良波史枝、林明子、佐藤文、樋野展正、柳沢達男、脇山素明、横山茂之
  • Organizer: 第10回日本蛋白質科学会年会
  • Place of Presentation: 札幌
  • Year and Date: 2010-06-17
• [Presentation] Escherichia coli translation elongation factor P (EF-P) is lysylated by GenX/PoxA/YjeA, a paralog of lysyl-tRNA synthetase, and YjeK (2010)
  • Author(s): Tatsuo Yanagisawa. Tomomi Sumida, Ryohei Ishii, Chie Takemoto, Shigeyuki Yokoyama
  • Organizer: Ribosomes2010
  • Place of Presentation: イタリア/オルビエート
• [Presentation] リジル-tRNA合成酵素パラログGenX/PoxA/YjeA、およびYjeKによる翻訳因子EF-Pの翻訳後修飾 (2010)
  • Author(s): 柳沢達男、澄田智美、石井亮平、竹本千重、横山茂之
  • Organizer: 日本RNA学会
  • Place of Presentation: 東京
• [Presentation] 大腸菌、昆虫細胞、動物細胞を用いた非天然型アミノ酸のタンパク質への部位特異的導入法の開発 (2010)
  • Author(s): 坂本健作、向井崇人、大竹和正、伊良波史枝、林明子、佐藤文、樋野展正、柳沢達男、脇山素明、横山茂之
  • Organizer: 日本蛋白質科学会
  • Place of Presentation: 札幌
• [Presentation] Structural bases of specific generation of amino-acid ligated tRNAs and proteins (2010)
  • Author(s): Shun-ichi Sekine、Takuhiro Ito、Tatsuo Yanagisawa、Shigeyuki Yokoyama
  • Organizer: 国際核酸化学シンポジウム (ISNAC-2010)
  • Place of Presentation: 横浜
• [Presentation] 翻訳後修飾アミノ酸のタンパク質への部位特異的導入 (2009)
  • Author(s): 坂本健作、小林隆嗣、柳沢達男、横山茂之
  • Organizer: 第4回無細胞生命科学研究会
  • Place of Presentation: 岐阜
  • Year and Date: 2009-11-16
• [Book] 化学と生物 (2011)
  • Author(s): 柳沢達男、澄田智美、横山茂之
  • Total Pages: 4
  • Publisher: 転移RNAを擬態する大腸菌翻訳因子EF-Pのアミノアシル化修飾-抗菌薬ターゲットとしての可能性に期待-
• [Book] Photon Factory Activity Report Research Highlights 2010 (2011)
  • Author(s): T. Yanagisawa, T. Sumida, S. Yokoyama
  • Total Pages: 1
  • Publisher: The Crystal Structure of Translation Elongation Factor P (EF-P) Complexed with An Aminoacyl-tRNA Synthetase Paralog, GenX, Reveals that EF-P Functionally Mimics Transfer RNA
• [Book] SPring-8 Research Frontiers 2010 (2011)
  • Author(s): T. Yanagisawa, T. Sumida, S. Yokoyama
  • Total Pages: 1
  • Publisher: Structure and function of a protein mimicking transfer RNA revealed from the structure of translation elongation factor P complexed with an aminoacyl-tRNA synthetase paralog, GenX.
• [Book] SPring-8 Research Frontiers 2010 : Structure and Function of a Protein Mimicking transfer RNA Revealed from the Structure of Translation Elongation Factor P Complexed with an Aminoacyl-tRNA Synthetase Paralog, GenX (2011)
  • Author(s): 柳沢達男、澄田智美、横山茂之
  • Total Pages: 2
  • Publisher: SPring-8
• [Book] ライフサイエンス新着論文レビュー:リジル-tRNA合成酵素パラログGenX-YjeKシステムによる翻訳因子EF-Pのリジル化修飾 (2010)
  • Author(s): 柳沢達男、横山茂之
  • Total Pages: 3
  • Publisher: ライフサイエンス統合データベースセンター
• [Book] RIKEN Research : Copycat protein finds a perfect match核酸と瓜二つのタンパク質 (2010)
  • Author(s): 柳沢達男、澄田智美、横山茂之
  • Total Pages: 1
  • Publisher: 理化学研究所
• [Patent(Industrial Property Rights)] 新規な非天然型アミノ酸、及びその利用(N^ε-(p-trifluoromethyldia zirinylbenzyloxycarbonyl)-lysine) (2011)
  • Inventor(s): 横山茂之、坂本健作、柳沢達男、樋野展正、伊良波史枝
  • Industrial Property Rights Holder: 横山茂之、坂本健作、柳沢達男、樋野展正、伊良波史枝
  • Industrial Property Rights Type: 特許
  • Filing Date: 2011-11-18
• [Patent(Industrial Property Rights)] 新規非天然型アミノ酸とその応用に関する研究: N(epsilin)-(p-trifluoromethyldiazirinylbenzylo xycarbonyl) lysine
  • Inventor(s): Yokoyama, S., Sakamoto, K., Yanagisawa, T., Hino, N., F. Iraha
  • Industrial Property Rights Holder: Yokoyama, S., Sakamoto, K., Yanagisawa, T., Hino, N., F. Iraha