Method for fluorescent labeling of proteins aiming at bio-imaging

• Project/Area Number: 19790094
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Drug development chemistry
• Research Institution: Nagoya City University
• Principal Investigator: UMEZAWA Naoki Nagoya City University, 大学院・薬学研究科, 准教授 (40347422)
• Project Period (FY): 2007 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥3,690,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥390,000); Fiscal Year 2008: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2007: ¥2,000,000 (Direct Cost: ¥2,000,000)
• Keywords: 蛍光標識 / タンパク質 / ペプチドタグ / ヒスタグ / 蛍光色素 / タンバク質

Research Abstract

本研究では、新規タンパク質蛍光ラベル化試薬を開発した。ヒスタグと呼ばれる短いペプチドと金属錯体との選択的かつ強固な結合を利用した。開発した試薬は、タンパク質との結合に伴い、蛍光強度が増大するという特長を持つ。同一の原理に基づき、紫外光励起可能なNTAC類と可視光励起可能なNTAF類の2群の新規試薬を開発した。これらの試薬は、ヒスタグを持つペプチド/タンパク質と選択的に結合し、蛍光強度が増大した。

Research Products

• [Journal Article] Turn-on fluorescent probe with visible light excitation for labeling of hexahistidine tagged protein (2009)
  • Author(s): M. Kamoto, N. Umezawa, N. Kato T. Higuchi
  • Journal Title: Bioorg. Med. Chem. Lett. 19 Pages: 2285-2288
  • Peer Reviewed
• [Journal Article] Extreme rate acceleration by axial thiolate coordination on the isomerization of endoperoxide catalyzed by iron porphyrin: relevance to prostaglandin H2 isomerase catalysis (2008)
  • Author(s): T. Yamane, K. Makino, N. Umezawa, N. Kato, and T. Higuchi
  • Journal Title: Angew. Chem. Int. Ed. 47 Pages: 6438-6440
  • Peer Reviewed
• [Journal Article] Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction (2008)
  • Author(s): S. Akita, N. Umezawa, N. Kato, and T. Higuchi
  • Journal Title: Bioorg. Med. Chem. 16 Pages: 7788-7794
  • Peer Reviewed
• [Journal Article] Novel probes showing specific fluorescence enhancement on binding to hexahistidine tag (2008)
  • Author(s): M. Kamoto, N. Umezawa, N. Kato, and T. Higuchi
  • Journal Title: Chem.-Eur. J. 14 Pages: 8004-8012
  • Peer Reviewed
• [Journal Article] Extreme rate acceleration by axial thiolate coordination on the isomerization of endoperoxide catalyzed by iron porphyrin: relevance to prostaglandin H_2 isomerase catalysis (2008)
  • Author(s): T. Yamane, K. Makino, N. Umezawa, N. Kato, T. Higuchi
  • Journal Title: Angew. Chem. Int. Ed. 47 Pages: 6438-6440
  • Peer Reviewed
• [Journal Article] Novel Probes Showing Specific Fluorescence Enhancement on Binding to Hexahistidine Tag (2008)
  • Author(s): M. Kamoto, N. Umezawa, N. Kato T. Higuchi
  • Journal Title: Chem.-Eur. J. 14 Pages: 8004-8012
  • Peer Reviewed
• [Journal Article] Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction (2008)
  • Author(s): S. Akita, N. Umezawa, N. Kato, T. Higuchi
  • Journal Title: Bioorg. Med. Chem. 16 Pages: 7788-7794
  • Peer Reviewed
• [Journal Article] Turn-on fluorescent probe with visible light excitation for labeling of hexahistidine tagged protein (2008)
  • Author(s): M. Kamoto, N. Umezawa, N. Ka to T. Higuchi
  • Journal Title: Bioorg. Med. Chem. Lett. 19 Pages: 2285-2288
  • Peer Reviewed
• [Journal Article] A versatile strategy for the synthesis of crown ether-bearing heterocycles : discovery of calcium-selective fluoroionophore (2007)
  • Author(s): Y. Aoki, N. Umezawa, Y. Asano, K. Hatano, Y. Yano, N. Kato, T. Higuchi
  • Journal Title: Bioorg. Med. Chem. 15 Pages: 7108-7115
  • Peer Reviewed
• [Journal Article] キナーゼ活性の蛍光検出 (2007)
  • Author(s): 梅澤直樹、樋口恒彦
  • Journal Title: 蛋白質核酸酵素 52 Pages: 1601-1607
• [Journal Article] アミノ酸、ペプチドの化学的性質を利用した生体高分子の選択的認識と検出 (2007)
  • Author(s): 梅澤直樹、秋田昌二、鴨東美絵、樋口恒彦
  • Journal Title: 薬学雑誌 127 Pages: 1915-1925
  • NAID: 110006546567
• [Journal Article] (α/β+α)-Peptide antagonists of BH3 domain/Bcl-XL recognition: toward generala atrategies for foldamer-based inhibition of protein -protein interactions (2007)
  • Author(s): J.D.Sadowsky, W.D.Fairlie, E.B.Hadley, H.-S.Lee, N.Um ezawa, Z.Nikolovska-Coleska, S.Wang, D.C.S.Huang, *Y.To mita, *S.H.Gellman
  • Journal Title: J. Am. Chem. Soc. 129 Pages: 139-154
  • Peer Reviewed
• [Journal Article] A versatile strategy for the synthesis of crown ether-bearing heterocycles: discovery of calcium-selective fluroionophore (2007)
  • Author(s): Y.Aoki, N.Umezawa, Y.Asano, K.Hatano, Y.Yano, N.Kato, *T.Higuchi
  • Journal Title: Bioorg. Med. Chem. 15 Pages: 7108-7115
  • Peer Reviewed
• [Journal Article] キナーゼ活性の蛍光検出 (2007)
  • Author(s): 梅澤 直樹、樋口 恒彦
  • Journal Title: 蛋白質 核酸 酵素 52 Pages: 1601-1607
• [Journal Article] アミノ酸、ペプチドの化学的性質を利用した生体高分子の選択的認識と検出 (2007)
  • Author(s): 梅澤 直樹、秋田 昌二、鴨東 美絵、樋口 恒彦
  • Journal Title: 薬学雑誌 127 Pages: 1915-1925
  • NAID: 110006546567
• [Presentation] ヒスタグ配列を認識する発蛍光型蛍光試薬の開発 (2009)
  • Author(s): 鴨東美絵、梅澤直樹、加藤信樹、樋口恒彦
  • Organizer: 日本薬学会第129年会
  • Place of Presentation: 京都
  • Year and Date: 2009-03-27
• [Presentation] ヒスタグ配列を認識する発蛍光型蛍光試薬の開発 (2009)
  • Author(s): 鴨東美絵、梅澤直樹、加藤信樹、樋口恒彦
  • Organizer: 日本薬学会第129年会
  • Place of Presentation: 国立京都国際会館(京都)
  • Year and Date: 2009-03-27
• [Presentation] ヒスタグペプチド配列を認識し発蛍光する蛍光試薬の開発 (2008)
  • Author(s): 鴨東美絵、梅澤直樹、加藤信樹、樋口恒彦
  • Organizer: 第3回バイオ関連合同シンポジウム
  • Place of Presentation: 東京工業大学すずかけ台キャンパス(神奈川)
  • Year and Date: 2008-09-18
• [Presentation] Novel probes showing specific fluorescence enhancement on binding to hexahistidine tag (2008)
  • Author(s): Mie Kamoto, Naoki Umezawa, Nobuki Kato, and Tsunehiko Higuchi
  • Organizer: 236th ACS National Meeting & Exposition
  • Place of Presentation: Philadelphia, USA
  • Year and Date: 2008-08-19
• [Presentation] Novel probes showing specific fluorescence enhancement on binding to hexahistidine tag (2008)
  • Author(s): M. Kamoto, N. Umezawa, N. Kato, T. Higuchi
  • Organizer: 236th ACS National Meeting & Exposition
  • Place of Presentation: Pennsylvania Convention Center (Philadelphia, USA)
  • Year and Date: 2008-08-19
• [Presentation] アミノ酸、ペプチドの化学的性質を利用した生体高分子の選択的認識と検出 (2008)
  • Author(s): 梅澤直樹
  • Organizer: 第41回若手ペプチド夏の勉強会
  • Place of Presentation: 京都
  • Year and Date: 2008-08-04
• [Presentation] アミノ酸、ペプチドの化学的性質を利用した生体高分子の選択的認識と検出 (2008)
  • Author(s): 梅澤直樹
  • Organizer: 第41回若手ペプチド夏の勉強会
  • Place of Presentation: プラムイン城陽(京都)
  • Year and Date: 2008-08-04
• [Presentation] Fluorescence assay for serine/threonine kinases using specific chemical reaction (2008)
  • Author(s): Naoki Umezawa, Shoji Akita, Nobuki Kato, and Tsunehiko Higuchi
  • Organizer: 236th ACS National Meeting & Exposition
  • Place of Presentation: Philadelphia, USA
• [Presentation] Fluorescence assay for serine/threonine kinases using specific chemical reaction (2008)
  • Author(s): N. Umezawa, S. Akita, N. Kato, T. Higuchi
  • Organizer: 236th ACS National Meeting & Exposition
  • Place of Presentation: Pennsylvania Convention Center (Philadelphia, USA)
• [Presentation] フォルダマーの可能性:蛋白-蛋白相互作用を阻害する分子を中心に (2007)
  • Author(s): 梅澤 直樹
  • Organizer: CBI学会第277回研究講演会
  • Place of Presentation: 東京
  • Year and Date: 2007-08-10
• [Presentation] Efficient fluorescence assay for serine/threonine kinases (2007)
  • Author(s): N. Umezawa
  • Organizer: AIMECS2007
  • Place of Presentation: Istanbul, Turkey
  • Year and Date: 2007-07-10
• [Presentation] Efficient fluorescence assay for serine/threonine kinases (2007)
  • Author(s): N.Umezawa
  • Organizer: AIMECS2007
  • Place of Presentation: Istanbul,Turky
  • Year and Date: 2007-07-10
• [Remarks]
  • URL: http://www.phar.nagoya-cu.ac.?jp/hp/ysk/index.html
• [Remarks]
  • URL: http://www.phar.nagoya-cu.ac.jp/research_course/rescourse02.html
• [Remarks]
  • URL: http://www.phar.nagoya-cu.ac.jp/hp/ysk/index.html
• [Remarks]
  • URL: http://www.phar.nagoya-cu.ac.jp/research_course/res_course02.html
• [Patent(Industrial Property Rights)] ペプチド又は蛋白質標識用の蛍光色素 (2008)
  • Inventor(s): 梅澤直樹、鴨東美絵、樋口恒彦
  • Industrial Property Rights Holder: 公立大学法人名古屋市立大学
  • Industrial Property Number: 2008-208933
  • Filing Date: 2008-08-14
• [Patent(Industrial Property Rights)] ケージド化合物の製造方法 (2007)
  • Inventor(s): 梅澤直雄、鵜飼和宏、樋口恒彦
  • Industrial Property Rights Holder: 公立大学法人名古屋市立大学
  • Filing Date: 2007-07-03
• [Patent(Industrial Property Rights)] マイケル反応による0n-chipリン酸化の検出方法 (2007)
  • Inventor(s): 梅澤直樹、鵜飼和宏、樋口恒彦
  • Industrial Property Rights Holder: 東洋紡績株式会社、公立大学法人名古屋市立大学
  • Filing Date: 2007-07-11