Analysis of protein fluctuation by introducing nonnatural amino acids

2012 Fiscal Year Final Research Report

• Project Area: Molecular Science of Fluctuations toward Biological Functions
• Project/Area Number: 20107005
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Japan Advanced Institute of Science and Technology
• Principal Investigator: HOHSAKA Takahiro 北陸先端科学技術大学院大学, マテリアルサイエンス研究科, 教授 (30263619)
• Project Period (FY): 2008 – 2012
• Keywords: 蛋白質 / 生物物理 / バイオテクノロジー / 非天然アミノ酸 / 蛍光 / 蛍光共鳴エネルギー移動 / 部位特異的変異導入

Research Abstract

In this study, an improved method for saturation mutagenesis allowing the substitution with natural and nonnatural amino acids to investigate the effect of the mutagenesis on proteins was developed. In addition, we developed a method for structural and functional protein analysis by introducing fluorescent nonnatural amino acids. Practical application of nonnatural amino acid-containing proteins as analytical tools and large-scale production of nonnatural proteins were also investigated.

Research Products

• [Journal Article] Synthesis of Novel BRET/FRET Protein Probes Containing Light-Emitting Proteins and Fluorescent Nonnatural Amino Acids (2012)
  • Author(s): Atsushi Yamaguchi, Takahiro Hohsaka
  • Journal Title: Bull. Chem. Soc. Jpn Volume: 85 Pages: 576-583
  • Peer Reviewed
• [Journal Article] Amber Codon-Mediated Expanded Saturation Mutagenesis of Proteins using a Cell-Free Translation System (2012)
  • Author(s): Naoki Shozen, Takayoshi Watanabe, Takahiro Hohsaka
  • Journal Title: J. Biosci. Bioeng Volume: 113 Pages: 704-709
  • Peer Reviewed
• [Journal Article] "Quenchbodies": Quench-based antibody probes that show antigen-dependent fluorescence. (2011)
  • Author(s): Ryoji Abe, Hiroyuki Ohashi, Issei Iijima, Masaki Ihara, Hiroaki Takagi, Takahiro Hohsaka, Hiroshi Ueda
  • Journal Title: J. Am. Chem. Soc Volume: 133 Pages: 17386-17394
  • Peer Reviewed
• [Journal Article] Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons (2010)
  • Author(s): Ryoji Abe, Kaori Shiraga, Shogo Ebisu, Hiroaki Takagi, Takahiro Hohsaka
  • Journal Title: J. Biosci. Bioeng Volume: 110 Pages: 32-38
  • Peer Reviewed
• [Journal Article] Position-specific incorporation of fluorescent non-natural amino acids into maltose-binding protein for detection of ligand binding by FRET and fluorescence quenching (2009)
  • Author(s): Issei Iijima, Takahiro Hohsaka
  • Journal Title: ChemBioChem Volume: 10 Pages: 999-1006
  • Peer Reviewed
• [Presentation] 非天然アミノ酸導入技術を利用したタンパク質の部位特異的PEG 化法の開発 (2013)
  • Author(s): 渡邉貴嘉、山口純、白神かおり、芳坂貴弘
  • Organizer: 日本化学会第93春季年会
  • Place of Presentation: 草津(滋賀)
  • Year and Date: 2013-03-24
• [Presentation] Site-specific incorporation of fluorescent nonnatural amino acids into proteins and its application to fluorescence analysis of proteins (2012)
  • Author(s): Takahiro Hohsaka
  • Organizer: 揺らぎが機能を決める生命分子の科学 第6回公開シンポジウム
  • Place of Presentation: 京都
  • Year and Date: 2012-12-05
• [Presentation] Application of nonnatural amino acid mutagenesis to proving protein fluctuations (2011)
  • Author(s): Takahiro Hohsaka
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 姫路(兵庫)
  • Year and Date: 2011-09-19
• [Presentation] 非天然アミノ酸のタンパク質への導入技術の開発とタンパク質の機能拡張への応用 (2011)
  • Author(s): 芳坂貴弘
  • Organizer: 第11回日本蛋白質科学会年会
  • Place of Presentation: 大阪
  • Year and Date: 2011-06-07
• [Presentation] FRET analysis of protein structures by double-incorporation of fluorescent and non-fluorescent nonnatural amino acids (2010)
  • Author(s): Issei Iijima, Takahiro Hohsaka
  • Organizer: PACIFICHEM2010
  • Place of Presentation: Honolulu, USA
  • Year and Date: 2010-12-17
• [Book] 「人工タンパク質の合成と応用」、超分子サイエンス&テクノロジー(国武豊喜監修) (2009)
  • Author(s): 芳坂貴弘
  • Total Pages: 890-895
  • Publisher: エヌティーエス
• [Remarks]
  • URL: http://www.jaist.ac.jp/ms/labs/hohsaka
• [Patent(Industrial Property Rights)] 芳香族アミノ基を有する非天然アミノ酸を導入したタンパク質の部位特異的な修飾方法 (2013)
  • Inventor(s): 芳坂貴弘
  • Industrial Property Rights Holder: 国立大学法人北陸先端科学技術大学院大学
  • Industrial Property Number: 出願、特願2013-019526
  • Filing Date: 2013-02-04