Site-specific incorporation of non-natural amino acids into proteins in living cells

2009 Fiscal Year Final Research Report

• Project/Area Number: 19380195
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied molecular and cellular biology
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: SAKAMOTO Kensaku The Institute of Physical and Chemical Research, 拡張遺伝暗号システム研究チーム, チームリーダー (50240685)
• Project Period (FY): 2007 – 2009
• Keywords: 非天然型アミノ酸 / 遺伝暗号 / タンパク質生合成 / 光クロスリンク / 翻訳後修飾 / 蛍光修飾 / 昆虫細胞

Research Abstract

I developed the technology for incorporating non-natural amino acids into proteins in living cells, and applied it for protein researches. Thus, (i) target proteins were labeled in mammalian cells, (ii) a naturally-occurring posttranslational modification was incorporated into protein at defined sites, (iii) novel protein-protein interactions were discovered by in vivo photo-crosslinking method, and (iv) non-natural amino acids were site-specifically incorporated into proteins in insect cells for the first time.

Research Products

• [Journal Article] Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr 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 (in press)
  • Peer Reviewed
• [Journal Article] Genetic encoding of non-natural amino acids in Drosophila melanogaster Schneider 2 cells (2010)
  • Author(s): Mukai, T., Wakiyama, M., Sakamoto, K., Yokoyama, S.
  • Journal Title: Protein Sci 19 Pages: 440-448
  • Peer Reviewed
• [Journal Article] Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography (2009)
  • Author(s): Sakamoto, K., Murayama, K., Oki, K., Iraha, F., Kato-Murayama, M., Takahashi, M., Ohtake, K., Kobayashi, T., Kuramitsu, S., Shirouzu, M., Yokoyama S.
  • Journal Title: Structure 17 Pages: 335-344
  • Peer Reviewed
• [Journal Article] Recognition of non-α-amino substrates by pyrrolysyl-tRNA synthetase (2009)
  • Author(s): Kobayashi, T., Yanagisawa, T., Sakamoto, K., Yokoyama, S.
  • Journal Title: J. Mol. Biol 385 Pages: 1352-1360
  • Peer Reviewed
• [Journal Article] ヨードチロシンの部位特異的導入と長波長X線を用いた単波長異常分散法による結晶構造解析 (2009)
  • Author(s): 村山和隆、坂本健作
  • Journal Title: 日本結晶学会誌 Vo.51 Pages: 251-257
• [Journal Article] Multi-step engineering of pyrrolysyl-tRNA synthetase to genetically encode N-(o-azidobenzyl oxycarbonyl)lysine for site-specific protein modification (2008)
  • Author(s): Yanagisawa, T., Ishii, R., Fukunaga, R., Kobayashi, T., Sakamoto, K., Yokoyama, S.
  • Journal Title: Chem. Biol 15 Pages: 1187-1197
  • Peer Reviewed
• [Journal Article] Transplantation of a tyrosine editing domain into a tyrosyl-tRNA synthetase variant enhances its specificity for a tyrosine analog (2008)
  • Author(s): Oki, K., Sakamoto, K., Kobayashi, T., Sasaki, H.M., Yokoyama, S.
  • Journal Title: Proc. Natl Acad. Sci. USA 105 Pages: 13298-13303
  • Peer Reviewed
• [Journal Article] Adding L-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases (2008)
  • Author(s): Mukai, T., Kobayashi, T., Hino, N., Yanagisawa, T., Sakamoto, K., Yokoyama, S.
  • Journal Title: Biochem. Biophys. Res. Commun 371 Pages: 818-822
  • Peer Reviewed
• [Journal Article] Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase (2008)
  • Author(s): Yanagisawa, T., Ishii, R., Fukunaga, R., Kobayashi, T., Sakamoto, K., Yokoyama, S.
  • Journal Title: J. Mol. Biol 378 Pages: 634-52
  • Peer Reviewed
• [Journal Article] Site-specific incorporation of non-natural amino acids into proteins in the mammalian cells with an expanded genetic code (2007)
  • Author(s): Hino, N., Hayashi, A., Sakamoto, K., Yokoyama, S.
  • Journal Title: Nature protocol 1 Pages: 2957-2962
  • Peer Reviewed
• [Journal Article] 非天然型アミノ酸をタンパク質へ部位特異的に導入する技術の開発とその応用 (2007)
  • Author(s): 坂本健作、横山茂之
  • Journal Title: 蛋白核酸酵素(共立出版) Vol.54,No.12 Pages: 65-71
• [Presentation] タンパク質複合体の光架橋による結晶構造解析技術の開発 (2009)
  • Author(s): 佐藤心、三桝信哉、佐藤文、樋野展正、坂本健作、梅原崇史、横山茂之
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 20091200
• [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): 小林隆嗣、坂本健作、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: 20091100
• [Presentation] 光クロスリンク法を用いた特定のモチーフを介するタンパク質間相互作用の網羅的解析 (2008)
  • Author(s): 樋野展正、尾山大明、佐藤文、向井崇人、秦裕子、山本雅、坂本健作、横山茂之
  • Organizer: 第31回日本分子生物学会年会・第81回日本生化学会合同大会(BMB2008)
  • Place of Presentation: 神戸
  • Year and Date: 20081200
• [Presentation] ヨードチロシンのタンパク質への部位特異的な導入と単波長異常分散法によるタンパク質結晶構造解析への応用 (2007)
  • Author(s): 坂本健作、大木健二、村山和隆、小林隆嗣、白水美香子、横山茂之
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会大会合同大会(BMB2007)
  • Place of Presentation: 横浜
  • Year and Date: 20071200
• [Presentation] 非天然型アミノ酸の導入によるタンパク質の部位特異的な蛍光標識技術 (2007)
  • Author(s): 伊良波史枝、向井崇人、佐藤文、林明子、樋野展正、保坂俊彰、山登一郎、大野敏、横川隆志、西川一八、坂本健作、横山茂之
  • Organizer: 第30回日本分子生物学会年会・第80回日本生化学会大会合同大会(BMB2007)
  • Place of Presentation: 横浜
  • Year and Date: 20071200
• [Remarks]
  • URL: http://protein.gsc.riken.go.jp/sakamoto/
• [Patent(Industrial Property Rights)] アミノアシルtrna合成酵素活性を有するポリペプチド及びその利用 (2008)
  • Inventor(s): 大木健二、坂本健作、横山茂之
  • Industrial Property Rights Holder: 理化学研究所
  • Industrial Property Number: 特許・特願2008-029236
  • Filing Date: 2008-02-08
• [Patent(Industrial Property Rights)] 変異体ピロリジル-trna合成酵素及びこれを用いる非天然アミノ酸組み込みタンパク質の製造方法 (2007)
  • Inventor(s): 小林隆嗣, 坂本健作, 横山茂之, 柳沢達男
  • Industrial Property Rights Holder: 理化学研究所
  • Industrial Property Number: 特許・特願2007-243574
  • Filing Date: 2007-09-20
• [Patent(Industrial Property Rights)] エステル結合を含む非天然タンパク質の製造方法
  • Inventor(s): 小林隆嗣, 向井崇人, 坂本健作, 横山茂之, 柳沢達男
  • Industrial Property Rights Holder: 理化学研究所
  • Industrial Property Number: 特許・特願2007-302452