Designing novel functions of proteins by using an artificial genetic code dictionary

• Project/Area Number: 20350075
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Chemistry related to living body
• Research Institution: Gifu University
• Principal Investigator: YOKOGAWA Takashi Gifu University, 工学部, 准教授 (90242304)
• Co-Investigator(Renkei-kenkyūsha): OHNO Satoshi 岐阜大学, 工学部, 助教 (10345796)
• Project Period (FY): 2008 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥19,890,000 (Direct Cost: ¥15,300,000、Indirect Cost: ¥4,590,000); Fiscal Year 2010: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2009: ¥7,410,000 (Direct Cost: ¥5,700,000、Indirect Cost: ¥1,710,000); Fiscal Year 2008: ¥8,450,000 (Direct Cost: ¥6,500,000、Indirect Cost: ¥1,950,000)
• Keywords: タンパク質合成系 / PURE システム / tRNA / 固相化プローブ法 / 遺伝暗号表 / 非天然アミノ酸 / Click chemistry / PUREシステム / ピロリシルtRNA合成酵素 / 最小tRNAセット / アグマチン / 分子整形術 / ピロリシン / Click Chemistry / M. acetivorans

Research Abstract

Restriction of the number of endogenous tRNAs in protein synthesis system causes "empty codons" in the genetic code dictionary. If we can create a tRNA that reads "an empty codon" and accepts an unnatural amino acid, it means that we develop an artificial genetic code dictionary. Here, I purified 21 tRNAs from Escherichia coli, and made a minimal tRNA set by remixing an individual tRNA. Actually, yellow fluorescent protein could be synthesized in PURE system with a minimal tRNA set and a chemically synthesized gene having minimal codons. It indicates that proteins having several unnatural amino acids can be effectively synthesized in the future.

Research Products

• [Journal Article] Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNA^<Tyr> pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion. (2010)
  • Author(s): Iraha, F., Oki, K., Kobayashi, T., Ohno, S., Yokogawa, T., Nishikawa, K., Yokoyama, S., Sakamoto, K.
  • Journal Title: Nucleic Acids Res. Vol.38, No.11 Pages: 3682-3691
  • Peer Reviewed
• [Journal Article] Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea. (2010)
  • Author(s): Ikeuchi, Y., Kimura, S., Numata, T., Nakamura, D., Yokogawa, T., Ogata, T., Wada, T., Suzuki, T., Suzuki, T.
  • Journal Title: Nat.Chem.Biol. Vol.6, No.4 Pages: 277-282
  • Peer Reviewed
• [Journal Article] Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts (2010)
  • Author(s): Yokogawa, T., Kitamura, Y., Nakamura, D., Ohno, S., Nishikawa, K.
  • Journal Title: Nucleic Acids Res. Vol.38, No.6 Pages: 89-89
  • Peer Reviewed
• [Journal Article] Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNA^<Tyr> pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion. (2010)
  • Author(s): Iraha, F., et al.
  • Journal Title: Nucleic Acids Res. Volume: 38 Pages: 3682-3691
  • Peer Reviewed
• [Journal Article] Agmatine-conjugated cytidine in a tRNA anticodon is essential for AUA decoding in archaea. (2010)
  • Author(s): Ikeuchi, Y., et al.
  • Journal Title: Nat.Chem.Biol. Volume: 6 Pages: 277-282
  • Peer Reviewed
• [Journal Article] N7-Methylguanine at position 46 (m^7G46) in rRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network. (2010)
  • Author(s): Tomikawa, C., et al.
  • Journal Title: Nucleic Acids Res. 38 Pages: 942-957
  • Peer Reviewed
• [Journal Article] Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. (2010)
  • Author(s): Yokogawa, T., et al.
  • Journal Title: Nucleic Acids Res. 38
  • Peer Reviewed
• [Journal Article] (2010)
  • Author(s): Yokogawa, T., et al.
  • Journal Title: Methods in Molecular Biology (Cell-Free Protein Production)(Humana Press) Pages: 227-242
• [Journal Article] Aquifex aeolicus tRNA (N2, N2-guanine)-dimethyltransfersae (Trml) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in tRNA. (2009)
  • Author(s): Awai, T., et al.
  • Journal Title: J.Biol.Chem. 284 Pages: 20467-20478
  • Peer Reviewed
• [Journal Article] Unconventional decoding of the AUA codon as methionine by mitochondrial tRNA^<Met> with the anticodon f5CAU as revealed with a mitochondrial in vitro translation system. (2009)
  • Author(s): Takemoto, C., et al.
  • Journal Title: Nucleic Acids Res. 37 Pages: 1616-1627
  • Peer Reviewed
• [Presentation] Methanosarcina acetivoransにおけるAUAコドン解読機構 (2010)
  • Author(s): 横川隆志
  • Organizer: 23回Archaea研究会
  • Place of Presentation: 名古屋
  • Year and Date: 2010-07-09
• [Presentation] Preparation of an ochre suppressor tRNA recognizing exclusively UAA codon by using the molecular surgery technique. (2009)
  • Author(s): 横川隆志
  • Organizer: 核酸化学シンポジウム
  • Place of Presentation: 高山
  • Year and Date: 2009-09-28
• [Presentation] Methanosarcina acetivoransPylRSを利用した部位特異的リシンアナログ導入タンパク質の合成 (2009)
  • Author(s): 横川隆志
  • Organizer: 22回Archaea研究会
  • Place of Presentation: 札幌
  • Year and Date: 2009-07-11
• [Presentation] Methanosarcina acetivorans PyIRSを利用した部位特異的リシンアナログ導入タンパク質の合成 (2009)
  • Author(s): 横川隆志
  • Organizer: 22回Archaea研究会
  • Place of Presentation: 札幌
  • Year and Date: 2009-07-11
• [Presentation] Methanosarcina acetivoransピロリシルtRNA合成酵素を利用したタンパク質の部位特異的修飾の試み (2008)
  • Author(s): 横川隆志
  • Organizer: 第10回RNAミーティング
  • Place of Presentation: 札幌
  • Year and Date: 2008-07-23
• [Presentation] Methanosarcina acetivoransの非標準アミノアシルtRNA合成酵素を利用した非標準アミノ酸含有タンパク質合成の試み (2008)
  • Author(s): 横川隆志
  • Organizer: 21回Archaea研究会
  • Place of Presentation: 沖縄
  • Year and Date: 2008-07-05
• [Book] Incorporation of 3-Azidotyrosine into Proteins Through Engineering Yeast Tyrosyl-tRNA Synthetase and Its Application to Site-Selective Protein Modification.(Cell-Free Protein Production) (2010)
  • Author(s): Yokogawa, T., Ohno, S., Nishikawa, K.
  • Publisher: Humana Press,Methods Mol. Biol.
• [Remarks] ホームページ等特に記載すべき事項なし