Development of sensitive multi-colour detection using the protein-protein interaction assay, FlimPIA

• Project/Area Number: 17K06920
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: Yuki Ohmuro 東京工業大学, 科学技術創成研究院, 助教 (30571088)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2019: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: タンパク質間相互作用 / 発光酵素 / タンパク質相互作用検出系 / タンパク質間相互作用検出 / 膜タンパク質 / 数理モデル / フォールディング / 立体障害 / 明るさ / 安定性 / タンパク質間相互作用検出系

Outline of Final Research Achievements

Protein-prottein interaction assay is a key technology to understanid life phenomena, make clear causes of diseases and create biosensors for detecting pathogenic bacterium and viruses.Therefore, Wide usages and user-friendly procedures are desireble for protein-protein interaction assay.
In this study, we improved the protein-protein interaction assay, FlimPIA developed previously, and FlimPIA became a significantly practical method.

Academic Significance and Societal Importance of the Research Achievements

FlimPIAにおけるプローブの熱安定性の向上：FlimPIAにおけるプローブの熱安定性を向上させることで、測定値の安定性が低く、またプローブの保存条件がやや厳しいという短所をなくす。
最小レポータータンパク質を利用したタンパク質間相互作用検出系の構築：ホタル由来発光酵素よりも小さなレポータータンパク質を利用することで、立体障害が小さく、プローブ作製が容易なタンパク質間相互作用検出系を構築する。

Research Products

• [Int'l Joint Research] ルイジアナ州立大学(米国)
• [Int'l Joint Research] ルイジアナ州立大学(米国)
• [Journal Article] Graphene Field Effect Transistor-Based Immunosensor for Ultrasensitive Noncompetitive Detection of Small Antigens (2020)
  • Author(s): Kanai Y, Ohmuro-Matsuyama Y, Tanioku M, Ushiba S, Ono T, Inoue K, Kitaguchi T, Kimura M, Ueda H, Matsumoto K
  • Journal Title: ACS Sens. Volume: 5 Issue: 1 Pages: 24-28
  • DOI: 10.1021/acssensors.9b02137
  • Peer Reviewed
• [Journal Article] Detection and Destruction of HER2-positive Cancer Cells by Ultra Quenchbody-siRNA Complex (2020)
  • Author(s): Dong J, Oka Y, Jeong HJ, Ohmuro-Matsuyama Y, Ueda H
  • Journal Title: Biotechnol. Bioeng. Volume: 117 Issue: 5 Pages: 1259-1269
  • DOI: 10.1002/bit.27302
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration With Analysis of Firefly Luciferase Mutants (2019)
  • Author(s): Dale R, Ohmuro-Matsuyama Y, Ueda H, Kato N
  • Journal Title: Biochmistry Volume: 58 Issue: 23 Pages: 2695-2702
  • DOI: 10.1021/acs.biochem.9b00272
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Creation of Stable and Strictly Regulated Enzyme Switch for Signal-On Immunodetection of Various Small Antigens (2019)
  • Author(s): Su J, Beh C, Ohmuro-Matsuyama Y, Kitaguchi T, Hoon S, Ueda H
  • Journal Title: J Biosci Bioeng. Volume: 128 Issue: 6 Pages: 677-682
  • DOI: 10.1016/j.jbiosc.2019.05.015
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Intrabody-based FRET probe to visualize endogenous histone acetylation (2019)
  • Author(s): Chung Chan-I、Sato Yuko、Ohmuro-Matsuyama Yuki、Machida Shinichi、Kurumizaka Hitoshi、Kimura Hiroshi、Ueda Hiroshi
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 10188-10188
  • DOI: 10.1038/s41598-019-46573-2
  • NAID: 120006936925
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Transmembrane Signaling on a Protocell: Creation of Receptor-Enzyme Chimeras for Immunodetection of Specific Antibodies and Antigens (2019)
  • Author(s): Su J, Kitaguchi T, Ohmuro-Matsuyama Y, Seah T, Ghadessy FJ, Hoon S, Ueda H
  • Journal Title: Sci Rep. Volume: 9 Issue: 1 Pages: 18189-18189
  • DOI: 10.1038/s41598-019-54539-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Evaluation of protein-ligand interactions using the luminescent interaction assay FlimPIA with streptavidin-biotin linkage. (2018)
  • Author(s): Ohmuro-Matsuyama Y, Yamashita T, Gomi K, Yamaji H, Ueda H.
  • Journal Title: Anal Biochem. Volume: 563 Pages: 61-66
  • DOI: 10.1016/j.ab.2018.10.010
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Homogeneous Noncompetitive Luminescent Immunodetection of Small Molecules by Ternary Protein Fragment Complementation (2018)
  • Author(s): 大室有紀・上田宏
  • Journal Title: Analytical Chemistry Volume: 90 Issue: 5 Pages: 3001-3004
  • DOI: 10.1021/acs.analchem.7b05140
  • Peer Reviewed
• [Journal Article] Improved sensitivity of firefly luminescent intermediate-based protein interaction assay using Ser 440 mutant with lower adenylation activity. (2017)
  • Author(s): 大室有紀・山下貴宏・LIn Huan・山地秀樹・上田宏
  • Journal Title: Luminescence Volume: 33 Issue: 1 Pages: 125-130
  • DOI: 10.1002/bio.3381
  • Peer Reviewed / Int'l Joint Research
• [Presentation] YFP断片融合抗体プローブを用いたヒストン翻訳後修飾検出系の構築 (2019)
  • Author(s): 大室（松山） 有紀, 北口 哲也, 木村 宏, 上田 宏
  • Organizer: 第71会生物工学会退会
• [Presentation] 分割YFP融合Intrabodyを用いたせい細胞内ヒストンアセチル化検出プローブの構築 (2019)
  • Author(s): 大室（松山） 有紀, 北口 哲也, 木村 宏, 上田 宏
  • Organizer: 第71回日本細胞生物学会大会 合同年次大会
• [Presentation] 小さく、高輝度・高発現なALucの作製 (2019)
  • Author(s): 大室（松山）有紀、金 誠培、上田 宏
  • Organizer: 生物発光化学発光研究会第35会学術講演会
• [Presentation] グラフェン電界効果トランジスタを利用したオープンサンドイッチ免疫測定法 (2019)
  • Author(s): 大室（松山）有紀、金井 康
  • Organizer: アライアンス若手交流会
  • Invited
• [Presentation] 発光酵素を利用した蛋白質間相互作用検出系・免疫測定法 (2019)
  • Author(s): 大室有紀・上田宏
  • Organizer: 第４回化学発光イメージングワークショップ
  • Invited
• [Presentation] Homogeneous noncompetitive immunological detection of small molecules by ternary luciferase fragment complementation (OS-BLIA (2018)
  • Author(s): Hiroshi Ueda, Yuki Ohmuro
  • Organizer: International Symposium on Bioluminescence and Chemiluminescence
  • Int'l Joint Research
• [Presentation] Mutant firefly luciferase with enhanced oxidative luminescent activity improved the response of protein-protein interaction assay FlimPIA (2018)
  • Author(s): Hiroshi Ueda, Yuki Ohmuro
  • Organizer: International Symposium on Bioluminescence and Chemiluminescence
  • Int'l Joint Research
• [Presentation] A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction (2018)
  • Author(s): Yuki Omuro, Hiroshi Ueda
  • Organizer: 第18回日本蛋白質科学会年会
  • Invited
• [Presentation] 発光酵素を用いたタンパク質相互作用検出系 (2018)
  • Author(s): 大室有紀
  • Organizer: LiHUB第5回創薬塾「創薬を目指した分子デザイン」
  • Invited
• [Presentation] 発光酵素を用いたタンパク質相互作用検出系FlimPIAとストレプトアビジンビーズを用いた相互作用検出系の構築 (2018)
  • Author(s): 大室有紀・五味恵子・山地秀樹・上田宏
  • Organizer: 生物発光化学発光研究会第34回学術講演会
• [Presentation] ストレプトアビジンビーズを用いたタンパク質間相互作用検出系FlimPIAの応用 (2018)
  • Author(s): 大室有紀・五味恵子・山地秀樹・上田宏
  • Organizer: 酵素工学研究会第80回講演会
• [Presentation] Development of ternary protein-protein interaction assay using peptide tags made from NanoLuc. (2018)
  • Author(s): 大室 有紀
  • Organizer: 第18回日本蛋白質科学会年会
  • Invited
• [Presentation] Mutant firefly luciferase with enhanced oxidative luminescent activity improved the response of protein-protein interaction assay FlimPIA (2018)
  • Author(s): 大室 有紀・上田 宏
  • Organizer: ISBC2018
  • Int'l Joint Research
• [Presentation] DEVELOPMENT OF RAPID IMMUNOASSSAY USING NANOLUC-DERIVED PEPTIDE TAGS (2017)
  • Author(s): 大室 有紀・上田 宏
  • Organizer: 生物発光化学発光研究会第33回学術講演会
• [Presentation] NanoLuc由来ペプチドタグを用いた簡便迅速な免疫測定法の開発 (2017)
  • Author(s): 大室有紀・蘇九龍・高橋里帆・上田宏
  • Organizer: 第69回日本生物工学会大会
• [Presentation] 蛋白質間相互作用検出系FlimPIAにおける発光値の向上 (2017)
  • Author(s): 大室有紀・上田宏
  • Organizer: 酵素工学研究会第78回講演会
• [Presentation] DEVELOPMENT OF RAPID IMMUNOASSSAY USING NANOLUC-DERIVED PEPTIDE TAGS (2017)
  • Author(s): 大室有紀・上田宏
  • Organizer: Enzyme Engineering XXIV
  • Int'l Joint Research
• [Presentation] Development of rapid bioluminescent immunoassay using Nanoluc-derived peptide tags (2017)
  • Author(s): 大室有紀・蘇九龍・上田宏
  • Organizer: 生物発光化学発光研究会第33回学術講演会
• [Book] Bioluminescence (2019)
  • Author(s): Ohmuro-Matsuyama Y, Ueda H
  • Total Pages: 16
  • Publisher: IntechOpen
• [Book] 発光イメージング実験ガイド (2019)
  • Author(s): 大室 有紀, 上田 宏
  • Total Pages: 6
  • Publisher: 羊土社
  • ISBN: 9784758122405
• [Book] Protein-Protein Interaction Assays (2018)
  • Author(s): Yuki Omuro, Hiroshi Ueda
  • Total Pages: 17
  • Publisher: IntechOpen
  • ISBN: 9781789233919
• [Book] Method in Molecular Biology (2017)
  • Author(s): 大室有紀・上田宏
  • Total Pages: 12
  • Publisher: SpringerLink
  • ISBN: 9781493969388
• [Remarks] 日経産業新聞 大室有紀 助教、上田宏 教授
  • URL: http://www.res.titech.ac.jp/news/award_release/release/20180313uk.html
• [Remarks] 極小サイズのペプチドタグで，蛋白質間相互作用を光で検出
  • URL: http://www.res.titech.ac.jp/news/reserch/20190312uk.html
• [Remarks] ３タンパク質断片相補による均相系での低分子の非競合発光免疫測定系の構築
  • URL: http://five-star.tagen.tohoku.ac.jp/results/distinguished_achievements.html
• [Remarks] FlimPIAについての新聞掲載
  • URL: http://www.res.titech.ac.jp/news/award_release/release/20180313uk.html
• [Remarks] FlimPIAについての新聞掲載
  • URL: http://www.res.titech.ac.jp/english/news/award_release/release/20180313uk.html
• [Remarks] FlimPIAの紹介
  • URL: https://atlasofscience.org/assaying-protein-protein-interaction-by-the-complementation-of-firefly-luciferase-catalytic-steps/
• [Patent(Industrial Property Rights)] バイオセンサー (2019)
  • Inventor(s): Su, Kitaguchi, Ohmuro et al.
  • Industrial Property Rights Holder: Su, Kitaguchi, Ohmuro et al.
  • Industrial Property Rights Type: 特許
  • Filing Date: 2019
  • Overseas