Development of New Strategy of Protein Design Based on Single Phage Sorting Technique

• Project/Area Number: 26282213
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Tohoku University
• Principal Investigator: TAKAHASHI Satoshi 東北大学, 多元物質科学研究所, 教授 (30283641)
• Co-Investigator(Kenkyū-buntansha): 坂本 清志 東北大学, 多元物質科学研究所, 助教 (30335228)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥16,770,000 (Direct Cost: ¥12,900,000、Indirect Cost: ¥3,870,000); Fiscal Year 2016: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2015: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2014: ¥7,150,000 (Direct Cost: ¥5,500,000、Indirect Cost: ¥1,650,000)
• Keywords: タンパク質デザイン / ファージ / 一分子蛍光観察 / ファージディスプレイ / 一分子ソーター / GFP / タンパク質 / 生物・生体工学 / 生物物理 / 一分子計測 / 蛍光タンパク質 / 一分子選別装置 / ファージディスプレイ実験 / 分割型GFP

Outline of Final Research Achievements

We planned to develop a new method of protein design by displaying a library of mutated fluorescent proteins on the surface of phages, and selecting phages displaying desired proteins by monitoring their emission properties. We constructed M13 phages displaying C-terminal fragment of GFP on g8p, and reconstituted GFP on the phage by adding the N-terminal fragment. We could create strongly emitting phages by displaying GFP on g8p proteins. We improved the sensitivity of the single phage detecting system. We were successful in selecting the fluorescent phages and examining the number of the collected phages by colony assay method.

Research Products

• [Journal Article] Significant Heterogeneity and Slow Dynamics of the Unfolded Ubiquitin Detected by the Line Confocal Method of Single-Molecule Fluorescence Spectroscopy (2016)
  • Author(s): Masataka Saito, Supawich Kamonprasertsuk, Satomi Suzuki, Kei Nanatani, Hiroyuki Oikawa, Keiichiro Kushiro, Madoka Takai, Po-ting Chen, Eric H.-L. Chen, Rita P.-Y. Chen, Satoshi Takahashi
  • Journal Title: J. Phys. Chem. B Volume: 120 Issue: 34 Pages: 8818-8829
  • DOI: 10.1021/acs.jpcb.6b05481
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Highly Collapsed Conformation of the Initial Folding Intermediates of β-Lactoglobulin with Non-Native α-Helix (2015)
  • Author(s): Tsuyoshi Konuma, Kazumasa Sakurai, Masanori Yagi, Yuji Goto, Teturo Fujisawa, Satoshi Takahashi
  • Journal Title: Journal of Molecular Biology Volume: 427 Issue: 19 Pages: 3158-3165
  • DOI: 10.1016/j.jmb.2015.07.018
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] One-dimensional sliding of p53 along DNA is accelerated in the presence of Ca(2+) or Mg(2+) at millimolar concentrations (2015)
  • Author(s): Murata A, Ito Y, Kashima R, Kanbayashi S, Nanatani K, Igarashi C, Okumura M, Inaba K, Tokino T, Takahashi S, Kamagata K
  • Journal Title: JOURNAL OF MOLECULAR BIOLOGY Volume: 427 Issue: 16 Pages: 2663-2678
  • DOI: 10.1016/j.jmb.2015.06.016
  • Peer Reviewed / Open Access
• [Journal Article] Complexity of the folding transition of the B domain of protein A revealed by the high-speed tracking of single-molecule fluorescence time series (2015)
  • Author(s): Hiroyuki Oikawa, Kiyoto Kamagata, Munehito Arai, Satoshi Takahashi
  • Journal Title: J. Phys. Chem. B Volume: 119 Issue: 20 Pages: 6081-6091
  • DOI: 10.1021/acs.jpcb.5b00414
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Where the complex things are: single molecule andensemble spectroscopic investigations of protein folding dynamics (2015)
  • Author(s): Satoshi Tkahahshi, Kiyoto Kamagata, Hiroyuki Oikawa
  • Journal Title: Current Opinion in Structural Biology Volume: 36 Pages: 1-9
  • DOI: 10.1016/j.sbi.2015.11.006
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Microsecond Dynamics of a Protein by Single-Molecule Fluorescence Measurements (2014)
  • Author(s): 小井川浩之、齊藤雅嵩、高橋聡
  • Journal Title: Seibutsu Butsuri Volume: 54 Issue: 5 Pages: 276-279
  • DOI: 10.2142/biophys.54.276
  • NAID: 130004693958
  • ISSN: 0582-4052, 1347-4219
  • Peer Reviewed / Open Access
• [Presentation] Construction of fluorescent phages based on split GFP for the phage sorting technique (2016)
  • Author(s): Naoki Mikoshiba, Yuuki Shimizu, Rie Kiriguchi, Seiji Sakamoto, Hiroyuki Oikawa, Kiyoto Kamagata, Takehiko Wada, Satoshi Takahashi
  • Organizer: 第５４回日本生物物理学会年会
  • Place of Presentation: 茨城県つくば市（つくば国際会議場）
  • Year and Date: 2016-11-25
• [Presentation] Development of a single-molecular sorting system based on fluorescence detection for protein design using phage display method (2016)
  • Author(s): 清水悠生、御子柴直紀、坂本清志、小井川浩之、鎌形清人、和田健彦、高橋聡
  • Organizer: 第５４回日本生物物理学会年会
  • Place of Presentation: 茨城県つくば市（つくば国際会議場）
  • Year and Date: 2016-11-25
• [Presentation] ユビキチンの折り畳みダイナミクスの一分子蛍光分光測定 (2015)
  • Author(s): 齊藤雅嵩, Chen Eric H.-L., Chen Po-Ting, Chen Rita P.-Y., 鎌形清人, 小井川浩之, 高橋聡
  • Organizer: 第9回分子科学討論会
  • Place of Presentation: 東京工業大学（東京都大田区）
  • Year and Date: 2015-09-16
• [Presentation] マイクロ秒分解一分子FRET測定よるタンパク質折り畳みダイナミクスの追跡 (2015)
  • Author(s): 小井川浩之, 新井宗仁, 深澤宏仁, 横田浩章, 井出徹, 高橋聡
  • Organizer: 第9回分子科学討論会
  • Place of Presentation: 東京工業大学（東京都大田区）
  • Year and Date: 2015-09-16
• [Presentation] 一分子蛍光分光法によって観測されたユビキチンの変性状態の不均一性 (2015)
  • Author(s): 齊藤雅嵩、Chen Eric H.-L., Chen Po-Ting, Chen Rita P.-Y., 鎌形清人, 小井川浩之, 高橋聡
  • Organizer: 第53回日本生物物理学会年会
  • Place of Presentation: 金沢大学（石川県金沢市）
  • Year and Date: 2015-09-13
• [Presentation] Tracking microsecond single-molecule FRET dynamics on the fast protein folding by the line-confocal microscopy (2015)
  • Author(s): Hiroyuki Oikawa, Munehito Arai, Atsuhito Fukasawa, Hiroaki Yokota , Toru Ide , Satoshi Takahashi
  • Organizer: 第53回日本生物物理学会年会
  • Place of Presentation: 金沢大学（石川県金沢市）
  • Year and Date: 2015-09-13
• [Presentation] 一分子蛍光分光法によるタンパク質のフォールディング研究 (2015)
  • Author(s): 高橋聡
  • Organizer: 機能物性融合科学研究会シリーズ２ソフトダイナミクス
  • Place of Presentation: 東京大学物性研究所（千葉県柏市）
  • Year and Date: 2015-04-02
  • Invited
• [Presentation] Conformation of the denatured BFP bound to GroEL by single molecule FRET measurements (2014)
  • Author(s): Aya Yoshida, Fumihiro Motojima, Hiroyuki OIkawa, Kiyoto Kamagata, Masasuke Yoshida and Satoshi Takahashi
  • Organizer: 第５２回日本生物物理学会年会
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Development of a new strategy of protein design: the single phage sorting based on fluorescence intensity (2014)
  • Author(s): Rie Kiriguchi, Toshihiko Kubota, Norihisa Takahashi, Seiji Sakamoto, Hiroyuki Oikawa, Kiyoto Kamagata , Takehiko Wada, Satoshi Takahashi
  • Organizer: 第５２回日本生物物理学会年会
  • Place of Presentation: 北海道札幌市
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] タンパク質フォールディングの分子科学 (2014)
  • Author(s): 高橋聡
  • Organizer: 第１回森野ディスカッション いつもそこには分子がある
  • Place of Presentation: 東京大学・小柴ホール
  • Year and Date: 2014-08-29
  • Invited
• [Presentation] Continuous tracking of protein folding at microsecond resolution by a line confocal detection of single molecule fluorescence (2014)
  • Author(s): Satoshi Takahashi
  • Organizer: The 28th Annual Symposium of The Protein Society
  • Place of Presentation: San Diego, USA
  • Year and Date: 2014-07-27 – 2014-07-30
  • Invited
• [Presentation] GroELに結合した基質BFPの一分子FRET計測による構造解析 (2014)
  • Author(s): 吉田文・元島史尋・田口英樹・小井川浩之・鎌形清人・吉田賢右・高橋聡
  • Organizer: 第14回日本蛋白質科学会年会
  • Place of Presentation: 神奈川県・横浜市
  • Year and Date: 2014-06-25 – 2014-06-27
• [Presentation] Microsecond-dynamics of the unfolded BdpA and ubiquitin by a line confocal detection of single molecule fluorescence (2014)
  • Author(s): Hiroyuki Oikawa , Masataka Saito, Po-Ting Chen, Hsin-Liang Chen, Rita P.-Y. Chen, Munehito Arai, Kiyoto Kamagata, Satoshi Takahashi
  • Organizer: The 4th Asia Pacific Protein Association Conference
  • Place of Presentation: Jeju, Korea
  • Year and Date: 2014-06-17