Utilization of microenvironment of nanotubes in in-vitro evolution

• Project/Area Number: 26350978
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: Kubo Tai 国立研究開発法人産業技術総合研究所, 創薬分子プロファイリング研究センター, 副研究センター長 (10178030)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2016: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2015: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2014: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: 試験管内進化 / マイクロ環境 / リフォールディング / 生理活性ペプチド / 分子進化 / 有機ナノチューブ

Outline of Final Research Achievements

We have found that organic nanotubes promoted refolding and thus leading innate activity of peptides when applied to the in-vitro translation reaction of the peptides. The observation was successfully adopted to the in-vitro evolution technology to construct a random peptide library with improved content of active peptides.

Research Products

• [Journal Article] Modulated dynamics of PAM-α7 nAChR from X-ray single molecular observations (2017)
  • Author(s): Tomoyuki Baba, Tai Kubo, Sumiko Ohashi, Keigo Ikezaki, Kazuhiro Mio, Hiroshi Sekiguchi, Yuji, C. Sasaki
  • Journal Title: BIOPHYSICAL JOURNAL Volume: 112-3 Pages: 327-327
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Random Peptide Library for Ligand and Drug Discovery (2016)
  • Author(s): Tai Kubo
  • Journal Title: Toxins and Drug Discovery Volume: － Pages: 1-24
  • DOI: 10.1007/978-94-007-6726-3_2-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A high performance platform based on cDNA display for efficient synthesis of protein fusions and accelerated directed evolution. (2016)
  • Author(s): Naimuddin, M., & Kubo, T.
  • Journal Title: ACC Combinatorial Science, Volume: 18 Pages: 117-129
  • DOI: 10.1021/acscombsci.5b00139
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Pr-SNTX, a short-chain three-finger toxin from Papuan pigmy mulga snake, is an antagonist of muscle-type nicotinic acetylcholine receptor (alpha2 beta delta epsilon) (2016)
  • Author(s): Yamauchi, Y., Kimoto, H., Yang, X., Filkin, S., Utkin, Y., Kubo, T., & Inagaki, H.
  • Journal Title: Biosci. Biotech. and Biochem Volume: 80 Pages: 158-161
  • DOI: 10.1080/09168451.2015.1065169
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Peptidome and transcriptome analysis of the toxin-like peptides in the venom glands of tarantula Grammostola rosea.” (2016)
  • Author(s): Kimura, T., & Kubo, T.
  • Journal Title: Spider Venoms, pp 251-270, Eds. P. Gopalakrishnakone, et al., Springer, Volume: - Pages: 251-270
  • DOI: 10.1007/978-94-007-6646-4_13-1
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Directed evolution of three-finger toxin to produce serine protease inhibitors (2014)
  • Author(s): Cai, W., Naimuddin, M., Inagaki, H., Kameyama, K., Ishida, N., & Kubo, T.*
  • Journal Title: J. Receptors and Signal Transduction Volume: 34 Pages: 154-161
  • Peer Reviewed
• [Journal Article] A versatile C-terminal specific biotinylation of proteins using both a puromycin-linker and a cell-free translation system for studying high-throughput protein-molecule interactions (2014)
  • Author(s): Nemoto N, Fukushima T, Kumachi S, Suzuki M, Nishigaki K, Kubo T.
  • Journal Title: Analytical Chemistry Volume: 86 Pages: 8535-8540
  • DOI: 10.1021/ac501601g
  • Peer Reviewed / Open Access
• [Journal Article] Real time ligand-induced motion mappings of AChBP and nAChR using X-ray single molecule tracking (2014)
  • Author(s): Sekiguchi, H., Suzuki, Y., Nishino, Y., Kobayashi, S., Shimoyama, Y., Cai, W., Nagata, K., Okada, M., Ichiyanagi, K., Ohta, N., Yagi, N., Miyazawa*, A., Kubo, T.*, & Sasaki, Y.*
  • Journal Title: Scientific Reports, Volume: -
  • DOI: 10.1038/srep06384
  • Peer Reviewed / Open Access
• [Presentation] Modulated dynamics of PAM-α7 nAChR from X-ray single molecular observations (2017)
  • Author(s): Tomoyuki Baba, Tai Kubo, Sumiko Ohashi, Keigo Ikezaki, Kazuhiro Mio, Hiroshi Sekiguchi, Yuji C. Sasaki
  • Organizer: Biophysical Society 61st Annual Meeting
  • Place of Presentation: New Orleans Ernest N. Morial Convention Center（米国ルイジアナ州ニューオリンズ）
  • Year and Date: 2017-02-14
  • Int'l Joint Research / Invited
• [Presentation] X線1分子追跡（DXT）法によるα7ニコチン性アセチルコリン受容体のリアルタイム1分子動態計測 (2016)
  • Author(s): 馬場知之、大橋澄子、関口博史、池崎圭吾、三尾和弘、佐々木裕次、久保泰
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-12-02
• [Presentation] リンカー設計およびタンパク質翻訳過程の至適化による試験管内進化技術（cDNAディスプレイ法）の改良と抗VEGF-3本指ペプチドの創製 (2016)
  • Author(s): 久保泰、Naimuddin Mohammed、多田耕平、大橋澄子、平家勇司、五島直樹
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-12-02
  • Invited
• [Presentation] 2種類の抗サバイビンバインダー（Three-FingerとVHH抗体）のサバイビン阻害活性 (2016)
  • Author(s): 安斎宏紀、鈴木武尊、四本勇也、木村慎之介、鈴木美穂、久保泰、根本直人
  • Organizer: 第39回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-11-30
• [Presentation] 3D Motion Maps of TRPV1 cation channel depicted by Diffracted X-Ray Tracking Method (2016)
  • Author(s): Kazuhiro Mio, Keigo, Ikezaki, Hiroshi Sekiguchi, Yufuku Matsushita, Tai Kubo, Yuji C. Sasaki
  • Organizer: 第54回生物物理学会
  • Place of Presentation: つくば国際会議場（茨城県つくば市）
  • Year and Date: 2016-11-27
• [Presentation] X-ray imaging of single protein's motion with ultra-high speed and accuracy (2016)
  • Author(s): Tomoyuki Baba, Keigo Ikezaki, Hiroshi Sekiguchi, Tai Kubo, Yuji C. Sasaki
  • Organizer: The 31st International Congress on High-speed Imaging and Photonics
  • Place of Presentation: ホテル阪急エキスポパーク（大阪府吹田市）
  • Year and Date: 2016-11-08
  • Int'l Joint Research
• [Presentation] Development of peptide neurotoxin-based allosteric modulators of nicotinic acetylcholine receptor alpha 7: evaluation by diffracted x-ray tracking method (2016)
  • Author(s): Tai Kubo, Tomoyuki Baba, Sumiko Ohashi, Keigo Ikezaki, Hiroshi Sekiguchi, Yuji C. Sasaki
  • Organizer: XII Congress of the Pan-America Section of the IST
  • Place of Presentation: Deauville Hotel（米国フロリダ州マイアミ）
  • Year and Date: 2016-09-18
  • Int'l Joint Research / Invited
• [Presentation] X線回折点追跡法と試験管内分子進化技術の融合によるチャネル分子の遷移過程識別プローブの創製 (2016)
  • Author(s): 久保泰
  • Organizer: 第68回埼玉大学脳科学セミナー
  • Place of Presentation: 埼玉大学（埼玉県さいたま市）
  • Year and Date: 2016-07-26
  • Invited
• [Presentation] イオンチャネルの遷移状態識別プローブ創製に向けて (2016)
  • Author(s): 久保泰
  • Organizer: 大阪大学蛋白質研究所セミナー
  • Place of Presentation: 大阪大学蛋白質研究所（大阪府豊中市）
  • Year and Date: 2016-05-12
  • Invited
• [Presentation] Realtime single molecular motion analysis of nicotinic acetylcholine receptor alpha 7 by diffracted X-ray tracking method (2016)
  • Author(s): Kubo, T., Baba, T., Ikezaki, K., Sekiguchi, H., Nishino, Y., Miyazawa, A., Sasaki, Y.C.
  • Organizer: 60th Annual Meeting of Biophysical Society
  • Place of Presentation: ロサンゼルス（米国）
  • Year and Date: 2016-02-29
  • Int'l Joint Research
• [Presentation] Utilization of neurotoxin-inspired peptide libraries in in-vitro evolution, and its proved pluripotency to target GPCRs, proteases and trophic factors (2015)
  • Author(s): Kubo, T., Naimuddin, M., Ono, S., Hirokawa, T.
  • Organizer: 18th World Congress of the International Society on Toxinology
  • Place of Presentation: オックスフォード（英国）
  • Year and Date: 2015-09-25
  • Int'l Joint Research
• [Presentation] In vitro evolution from pluripotent peptide libraries with natural neurotoxin scaffolds to target receptors, proteases and trophic factors (2015)
  • Author(s): Kubo, T., Naimuddin, M., Ono, S.
  • Organizer: 29th Annual Symposium of the Protein Society
  • Place of Presentation: バルセロナ（スペイン）
  • Year and Date: 2015-07-23
  • Int'l Joint Research
• [Presentation] Pluripotent random peptide library inspired by natural neurotoxins in the accelerated gene evolution (2015)
  • Author(s): Kubo, T., Naimuddin, M., Ono, S.
  • Organizer: 10th European Biophysics Congress
  • Place of Presentation: ドレスデン （ドイツ）
  • Year and Date: 2015-07-18
  • Int'l Joint Research
• [Presentation] パプアピグミーマルガスネークの生理活性ペプチドおよびタンパク質に関する研究 (2015)
  • Author(s): 山内瑶子、木本 光、鳥羽通久、久保 泰、稲垣 英利
  • Organizer: 第６２回トキシンシンポジウム
  • Place of Presentation: 志摩（三重）
  • Year and Date: 2015-07-10
• [Presentation] 加速進化型ペプチドのスキャフォールドを利用した分子認識 (2015)
  • Author(s): 久保泰
  • Organizer: 人工認識分子研究会
  • Place of Presentation: 大阪（大阪）
  • Year and Date: 2015-06-23
  • Invited
• [Presentation] 加速進化型ペプチドの分子骨格を利用した多能性ペプチドライブラリの利用技術 (2015)
  • Author(s): 久保泰
  • Organizer: 第20回分子複合医薬研究会
  • Place of Presentation: 池田
  • Year and Date: 2015-02-26
  • Invited
• [Presentation] 加速進化型ペプチドのscaffoldに基づく多能性ペプチドライブラリの構築と標的特異的分子の創製 (2014)
  • Author(s): 久保泰
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2014-11-26
• [Presentation] 高速1分子内ダイナミクス計測で捉えるアセチルコリン受容体・機能運動 (2014)
  • Author(s): 関口博史、徳江真紀、西野有里、一柳光平、久保泰、宮澤淳夫、八木直人、佐々木裕次
  • Organizer: 第14回日本蛋白質科学会
  • Place of Presentation: 横浜
  • Year and Date: 2014-06-25 – 2014-06-27
• [Presentation] Utilization of three-finger peptide library and evolution in vitro toward protease inhibitors. (2014)
  • Author(s): Cai, W., Kubo, T.
  • Organizer: 10th IST Asia Pacific Congress on Animal, Plant and Microbial Toxins
  • Place of Presentation: Changsha, China
  • Year and Date: 2014-06-16