Novel membrane proteins by combining computational design and in vitro directed eovlution

• Project/Area Number: 17H00888
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Tokyo Institute of Technology (2020-2021); Osaka University (2017-2019)
• Principal Investigator: Matsuura Tomoaki 東京工業大学, 地球生命研究所, 教授 (50362653)
• Co-Investigator(Kenkyū-buntansha): 大崎 寿久 地方独立行政法人神奈川県立産業技術総合研究所, 人工細胞膜システムグループ, サブリーダー (50533650) ; 藤井 聡志 地方独立行政法人神奈川県立産業技術総合研究所, 人工細胞膜システムグループ, 研究員 (60619005)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥43,160,000 (Direct Cost: ¥33,200,000、Indirect Cost: ¥9,960,000); Fiscal Year 2020: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000); Fiscal Year 2019: ¥9,100,000 (Direct Cost: ¥7,000,000、Indirect Cost: ¥2,100,000); Fiscal Year 2018: ¥9,620,000 (Direct Cost: ¥7,400,000、Indirect Cost: ¥2,220,000); Fiscal Year 2017: ¥15,340,000 (Direct Cost: ¥11,800,000、Indirect Cost: ¥3,540,000)
• Keywords: 膜タンパク質 / リポソーム / 進化分子工学 / 人工細胞 / 無細胞タンパク質合成系 / デノボデザイン / 人工膜タンパク質 / バイオテクノロジー / リポソームディスプレイ法 / 無細胞翻訳系 / 合理的設計 / 生物物理

Outline of Final Research Achievements

Only a small fraction of the sequence space has been explored by nature through evolution. By rational design, it is now clear that for soluble proteins, there are many artificial proteins that possess structures and functions in the unexplored sequence space. In this study, we show that there are molecules with artificial membrane proteins that also have functions and structures in membrane proteins. Specifically, we designed an artificial membrane protein with a 2 nm pore and clarified its function using an artificial cell reactor.

Academic Significance and Societal Importance of the Research Achievements

人工細胞を構築する研究は、非生命から生命をどのように創り出せるかを明らかにする方法の一つである。現在までに、細胞の機能を模倣した様々な人工細胞が構築されてきた。本研究課題では、人工細胞が人工膜タンパク質の機能解析に用いること、更には、膜タンパク質進化分子工学に使えることを示すものである。

Research Products

• [Int'l Joint Research] University of Washington(米国)
• [Int'l Joint Research] University of Washington/Stanford University(米国)
• [Int'l Joint Research] University of Washington(米国)
• [Int'l Joint Research] Stanford University/University of Washington(米国)
• [Journal Article] Identification of conditions for efficient cell-sized liposome preparation using commercially available reconstituted in vitro transcription-translation system (2022)
  • Author(s): Uyeda, A.; Reyes, S. G.; Kanamori, T.; Matsuura, T.
  • Journal Title: J Biosci Bioeng Volume: 133 (2) Issue: 2 Pages: 181-186
  • DOI: 10.1016/j.jbiosc.2021.10.008
  • NAID: 210000159461
  • Peer Reviewed
• [Journal Article] Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display (2022)
  • Author(s): Nakai Hiroki、Isshiki Kinuka、Hattori Masato、Maehira Hiromasa、Yamaguchi Tatsumi、Masuda Keiko、Shimizu Yoshihiro、Watanabe Takayoshi、Hohsaka Takahiro、Shihoya Wataru、Nureki Osamu、Kato Yasuhiko、Watanabe Hajime、Matsuura Tomoaki
  • Journal Title: Analytical Chemistry Volume: 94 Issue: 9 Pages: 3831-3839
  • DOI: 10.1021/acs.analchem.1c04714
  • Peer Reviewed
• [Journal Article] Chimeric mutants of staphylococcal hemolysin, which act as both one‐component and two‐component hemolysin, created by grafting the stem domain (2022)
  • Author(s): Ghanem Nouran、Kanagami Natsuki、Matsui Takashi、Takeda Kein、Kaneko Jun、Shiraishi Yasuyuki、Choe Christian A.、Uchikubo‐Kamo Tomomi、Shirouzu Mikako、Hashimoto Tsubasa、Ogawa Tomohisa、Matsuura Tomoaki、Huang Po‐Ssu、Yokoyama Takeshi、Tanaka Yoshikazu
  • Journal Title: The FEBS Journal Volume: 11 Issue: 12 Pages: 11-20
  • DOI: 10.1111/febs.16354
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Computational design of transmembrane pores. (2020)
  • Author(s): Xu, C.; Lu, P.; Gamal El-Din, T. M.; Pei, X. Y.; Johnson, M. C.; Uyeda, A.; Bick, M. J.; Xu, Q.; Jiang, D.; Bai, H.; Reggiano, G.; Hsia, Y.; Brunette, T. J.; Dou, J.; Ma, D.; Lynch, E. M.; Boyken, S. E.; Huang, P. S.; Stewart, L.; DiMaio, F.; Kollman, J. M.; Luisi, B. F.; Matsuura, T.; Catterall, W. A.; Baker, D.
  • Journal Title: Nature Volume: 585 (7823) Issue: 7823 Pages: 129-134
  • DOI: 10.1038/s41586-020-2646-5
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Programmable Artificial Cells Using Histamine-Responsive Synthetic Riboswitch (2019)
  • Author(s): Dwidar Mohammed、Seike Yusuke、Kobori Shungo、Whitaker Charles、Matsuura Tomoaki、Yokobayashi Yohei
  • Journal Title: Journal of the American Chemical Society Volume: 141 Issue: 28 Pages: 11103-11114
  • DOI: 10.1021/jacs.9b03300
  • Peer Reviewed / Open Access
• [Journal Article] Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA (2019)
  • Author(s): Noba Kosaku、Ishikawa Masahito、Uyeda Atsuko、Watanabe Takayoshi、Hohsaka Takahiro、Yoshimoto Shogo、Matsuura Tomoaki、Hori Katsutoshi
  • Journal Title: Journal of the American Chemical Society Volume: 141 Issue: 48 Pages: 19058-19066
  • DOI: 10.1021/jacs.9b09340
  • Peer Reviewed
• [Journal Article] Class?III Polyphosphate Kinase?2 Enzymes Catalyze the Pyrophosphorylation of Adenosine‐5′‐Monophosphate (2019)
  • Author(s): Ogawa Marin、Uyeda Atsuko、Harada Kazuo、Sato Yu、Kato Yasuhiko、Watanabe Hajime、Honda Kohsuke、Matsuura Tomoaki
  • Journal Title: ChemBioChem Volume: 20 Issue: 23 Pages: 2961-2967
  • DOI: 10.1002/cbic.201900303
  • Peer Reviewed / Open Access
• [Journal Article] In vitro synthesis of the human calcium transporter Letm1 within cell-sized liposomes and investigation of its lipid dependency (2019)
  • Author(s): Okamura Kosuke、Matsushita Shuhei、Kato Yasuhiko、Watanabe Hajime、Matsui Aiko、Oka Toshihiko、Matsuura Tomoaki
  • Journal Title: Journal of Bioscience and Bioengineering Volume: 127 Issue: 5 Pages: 544-548
  • DOI: 10.1016/j.jbiosc.2018.11.003
  • NAID: 40021891579
  • Peer Reviewed / Open Access
• [Journal Article] Quantitative analysis of cell-free synthesized membrane proteins at the stabilized droplet interface bilayer (2018)
  • Author(s): Elfaramawy Maie A.、Fujii Satoshi、Uyeda Atsuko、Osaki Toshihisa、Takeuchi Shoji、Kato Yasuhiko、Watanabe Hajime、Matsuura Tomoaki
  • Journal Title: Chemical Communications Volume: 54 Issue: 86 Pages: 12226-12229
  • DOI: 10.1039/c8cc06804f
  • Peer Reviewed
• [Journal Article] Concurrent In Vitro Synthesis and Functional Detection of Nascent Activity of the KcsA Channel under a Membrane Potential (2018)
  • Author(s): Iwamoto M, Elfaramawy MA, Yamatake M, Matsuura T, Oiki S
  • Journal Title: ACS Synthetic Biology Volume: 7 Issue: 4 Pages: 1004-1011
  • DOI: 10.1021/acssynbio.7b00454
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Robustness of a Reconstituted Escherichia coli Protein Translation System Analyzed by Computational Modeling (2018)
  • Author(s): Matsuura Tomoaki、Hosoda Kazufumi、Shimizu Yoshihiro
  • Journal Title: ACS Synthetic Biology Volume: 7 Issue: 8 Pages: 1964-1972
  • DOI: 10.1021/acssynbio.8b00228
  • Peer Reviewed
• [Journal Article] Different protein localizations on the inner and outer leaflet of cell-sized liposomes using cell-free protein synthesis (2018)
  • Author(s): Uyeda Atsuko、Watanabe Takayoshi、Hohsaka Takahiro、Matsuura Tomoaki
  • Journal Title: Synthetic Biology Volume: 3 Issue: 1
  • DOI: 10.1093/synbio/ysy007
  • Peer Reviewed
• [Presentation] 要素から創る無細胞分子システムの構築と応用 (2022)
  • Author(s): 松浦友亮
  • Organizer: 日本ケミカルバイオロジー学会 産学連携委員会
  • Invited
• [Presentation] Bottom-up construction of synthetic cells and its relation to Earth-Life science (2022)
  • Author(s): Tomoaki Matsuura
  • Organizer: 10th ELSI symposium (Online organized by ELSI, Tokyo Tech)
  • Int'l Joint Research / Invited
• [Presentation] In vitro directed evolution of membrane proteins using ribosome and liposome display (2021)
  • Author(s): Tomoaki Matsuura
  • Organizer: New Proteins by Evolution and Engineering (Online organized by OIST)
  • Int'l Joint Research / Invited
• [Presentation] Engineering and characterizing membrane proteins using artificial cells (2019)
  • Author(s): Tomoaki Matsuura
  • Organizer: New Frontier in Protein Design & Engineering
  • Int'l Joint Research / Invited
• [Presentation] Evolutionary engineering and characterization of membrane proteins using liposome display (2018)
  • Author(s): Tomoaki Matsuura
  • Organizer: Protein Engineering Canada 2018
  • Int'l Joint Research / Invited
• [Presentation] 人工細胞リアクターの創生と応用 (2018)
  • Author(s): 松浦友亮
  • Organizer: 白鷺セミナー
  • Invited
• [Presentation] 再構成型セルフリータンパク質合成系を用いたタンパク質配列空間と成分濃度空間探索 (2018)
  • Author(s): 松浦友亮
  • Organizer: 2018年度日本農芸化学会
  • Invited
• [Presentation] リポソームディスプレイ法を用いた膜タンパク質の創生と解析 (2017)
  • Author(s): 松浦友亮
  • Organizer: 2017年度生命科学系学会合同年次大会（ConBio2017）
  • Invited
• [Presentation] Decorating liposome surface with biological molecule and its application (2017)
  • Author(s): Tomoaki Matsuura
  • Organizer: IGER International Symposium on Cell Surface Structures and Functions 2017
  • Int'l Joint Research / Invited
• [Presentation] In vitro membrane protein engineering by liposome display (2017)
  • Author(s): Tomoaki Matsuura
  • Organizer: The 17th KIAS Conference on Protein Structure and Function
  • Int'l Joint Research / Invited