Directed evolution of membrane proteins using cell-free protein synthesis system

• Project/Area Number: 25282239
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Chemical biology
• Research Institution: Osaka University
• Principal Investigator: Matsuura Tomoaki 大阪大学, 工学(系)研究科(研究院), 准教授 (50362653)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000); Fiscal Year 2015: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2014: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2013: ¥12,350,000 (Direct Cost: ¥9,500,000、Indirect Cost: ¥2,850,000)
• Keywords: 進化分子工学 / 無細胞翻訳系 / 膜タンパク質 / リポソーム / 膜蛋白質

Outline of Final Research Achievements

Liposome display is an evolutionary method that enables the directed evolution of membrane proteins in vitro. The method is based on the syntheses of membrane proteins using an in vitro transcription–translation system (IVTT) inside cell-sized phospholipid vesicles. Here, using EmrE, a multidrug transporter from Escherichia coli, as a model protein, the screening was performed based on two functions of EmrE: substrate transport activity and membrane integration activity. Starting from a mock gene library prepared by mixing an active and an inactive gene, 10- to 35-fold enrichment of the active genes was obtained. In addition, from a random mutagenized library of EmrE, gene pool exhibiting higher activity than the wild-type was obtained. We also succeeded in introducing the membrane protein transport machinery to the liposome display method, which expanded the repertories of the membrane protein that can be applied to liposome display.

Research Products

• [Journal Article] Liposome-based in vitro evolution of aminoacyl-tRNA synthetase for enhanced pyrrolysine derivative incorporation. (2015)
  • Author(s): Uyeda A, Watanabe T, Kato Y, Watanabe H, Yomo T, Hohsaka T, Matsuura T
  • Journal Title: Chembiochem Volume: 16 Issue: 12 Pages: 1797-1802
  • DOI: 10.1002/cbic.201500174
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] <i>In vitro</i> directed evolution of alpha-hemolysin by liposome display (2015)
  • Author(s): Fujii, S., Matsuura, T., and Yomo, T.
  • Journal Title: BIOPHYSICS Volume: 11 Issue: 0 Pages: 67-72
  • DOI: 10.2142/biophysics.11.67
  • NAID: 130004940815
  • ISSN: 1349-2942
  • Peer Reviewed / Open Access
• [Journal Article] MembranCurvature Affects the Formation of alpha-Hemolysin Nanopores (2015)
  • Author(s): Fujii, S., Matsuura, T., and Yomo, T.
  • Journal Title: ACS Chem Biol. Volume: 未定 Issue: 7 Pages: 1694-701
  • DOI: 10.1021/acschembio.5b00107
  • Peer Reviewed
• [Journal Article] Effects of ribosomes on the kinetics of Qb replication (2014)
  • Author(s): Usui, K., Ichihashi, N., Kazuta, Y., Matsuura, T., Yomo, T
  • Journal Title: FEBS Letters Volume: 588 Issue: 1 Pages: 117-123
  • DOI: 10.1016/j.febslet.2013.11.018
  • Peer Reviewed
• [Journal Article] The evolutionary enhancement of genotype-phenotype linkages in the presence of multiple copies of genetic material (2014)
  • Author(s): Uno, K., Sunami, T., Ichihashi, N., Kazuta, Y., Matsuura, T., and Yomo, T.
  • Journal Title: Chembiochem Volume: 15 Issue: 15 Pages: 2291-2288
  • DOI: 10.1002/cbic.201402299
  • Peer Reviewed
• [Journal Article] Identification of giant unilamellar vesicles with permeability to small charged molecules (2014)
  • Author(s): Nishimura, K., Matsuura, T., Sunami, T., Fujii, S., Nishimura, K., Suzuki, H., and Yomo, T.
  • Journal Title: Rsc Adv Volume: 4(66) Issue: 66 Pages: 35224-35232
  • DOI: 10.1039/c4ra05332j
  • Peer Reviewed
• [Journal Article] Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system (2014)
  • Author(s): Kazuta, Y., Matsuura, T., Ichihashi, N., and Yomo, T.
  • Journal Title: J Biosci Bioeng Volume: 118 Issue: 5 Pages: 1578-1591
  • DOI: 10.1016/j.jbiosc.2014.04.019
  • NAID: 110009894841
  • Peer Reviewed
• [Journal Article] Liposome display for in vitro selection and evolution of membrane proteins (2014)
  • Author(s): Fujii, S., Matsuura, T., Sunami, T., Nishikawa, T., Kazuta, Y., and Yomo, T.
  • Journal Title: Nat Protoc Volume: 9 Issue: 7 Pages: 1578-1591
  • DOI: 10.1038/nprot.2014.107
  • Peer Reviewed
• [Journal Article] In Vitro Membrane Protein Synthesis Inside Cell-Sized Vesicles Reveals the Dependence of Membrane Protein Integration on Vesicle Volume. (2013)
  • Author(s): Soga, H., Fujii, S., Yomo, T., Kato, Y., Watanabe, H. and Matsuura, T.
  • Journal Title: ACS synthetic biology Volume: 0 Issue: 6 Pages: 372-379
  • DOI: 10.1021/sb400094c
  • Peer Reviewed / Open Access
• [Journal Article] Cell-free Protein Synthesis in a Microchamber Revealed the Presence of an Optimum Compartment Volume for High-order Reactions (2013)
  • Author(s): T. Okano, T. Matsuura, H. Suzuki, and T. Yomo
  • Journal Title: ACS Synthetic Biology Volume: 3 Issue: 6 Pages: 347-352
  • DOI: 10.1021/sb400087e
  • Peer Reviewed / Open Access
• [Journal Article] Kinetic model of double-stranded RNA formation during long RNA replication by Qβreplicase (2013)
  • Author(s): Usui, K., Ichihashi, N., Kazuta, Y., Matsuura, T., Yomo, T
  • Journal Title: FEBS Letters Volume: 587 Issue: 16 Pages: 2565-2571
  • DOI: 10.1016/j.febslet.2013.06.033
  • Peer Reviewed
• [Journal Article] Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment (2013)
  • Author(s): Ichihashi, N., Usui, K., Kazuta, Y., Sunami, T., Matsuura T., Yomo, T
  • Journal Title: Nature Communications Volume: 4 Issue: 1 Pages: 1-7
  • DOI: 10.1038/ncomms3494
  • NAID: 120006831817
  • Peer Reviewed
• [Presentation] 無細胞タンパク質合成系を用いた膜タンパク質進化分子工学 (2016)
  • Author(s): 松浦友亮
  • Organizer: 第4回 ネオバイオ分子研究会
  • Place of Presentation: 大阪府立大学I-site なんば・大阪市
  • Year and Date: 2016-01-29
  • Invited
• [Presentation] バイオ界面を利用したin vitro進化分子工学 (2015)
  • Author(s): 松浦友亮
  • Organizer: 日本生物工学会第67回大会
  • Place of Presentation: 城山観光ホテル・鹿児島市
  • Year and Date: 2015-10-26
  • Invited
• [Presentation] 無細胞タンパク質合成系を用いた膜タンパク質進化分子工学 (2015)
  • Author(s): 松浦友亮
  • Organizer: 第15回日本蛋白質科学会年会
  • Place of Presentation: あわぎんホール, 徳島市
  • Year and Date: 2015-06-24
  • Invited
• [Presentation] In Vitro Selection and Evolution of Membrane Proteins” Gordon Research Conferences in Proteins (2015)
  • Author(s): Tomoaki Matsuura
  • Organizer: Gordon Research Conferences in Proteins
  • Place of Presentation: Boston, U.S.A.
  • Year and Date: 2015-06-15
  • Int'l Joint Research / Invited
• [Presentation] in vitroで生体分子を使って組み上げる生命システム. (2014)
  • Author(s): 松浦友亮
  • Organizer: 国際高等研研究プロジェクト「分子基盤に基づく生体機能ネットワークとダイナミクスの解明」第1回研究会プログラム
  • Place of Presentation: 国際高等研研究（木津川市）
  • Year and Date: 2014-12-14 – 2014-12-15
  • Invited
• [Presentation] IN VITRO EVOLUTION OF alpha-HEMOLYSIN USING A LIPOSOME DISPLAY (2014)
  • Author(s): Tomoaki Matsuura
  • Organizer: Protein Society Meeting
  • Place of Presentation: SanDiego, U. S. A.
  • Year and Date: 2014-07-27 – 2014-07-30
  • Invited
• [Presentation] Engineering Membrane Proteins Entirely in vitro with Liposome Display Technology (2014)
  • Author(s): Tomoaki Matsuura
  • Organizer: KSBB SPRING MEETING and INTERNATIONAL SYMPOSIUM
  • Place of Presentation: Hyundai Hotel, Gyeongju, Korea
  • Year and Date: 2014-04-10
  • Invited
• [Presentation] 無細胞系を用いたタンパク質エンジニアリングの新技術 (2014)
  • Author(s): 松浦友亮
  • Organizer: 2013年度日本生物工学会技術セミナー
  • Place of Presentation: 神戸大学瀧川記念学術交流会館
  • Invited
• [Presentation] In vitro evolution and selection of membrane proteins with liposome display technology (2014)
  • Author(s): 松浦友亮
  • Organizer: Korea-Japan Smart Biodesign Workshop: Technology exchange for green biotechnology
  • Place of Presentation: Aobayama Memorial Hall, Tohoku University
  • Invited
• [Presentation] Chemistry of “Origins of life” (2014)
  • Author(s): 松浦友亮
  • Organizer: JFFos Symposium
  • Place of Presentation: Metz,France
  • Invited
• [Presentation] In vitro evolution of alpha-hemolysin using a liposome display (2013)
  • Author(s): 松浦友亮
  • Organizer: 第22回酵素工学国際会議
  • Place of Presentation: 富山国際会議場
• [Presentation] タンパク質合成反応シミュレーターの構築 (2013)
  • Author(s): 松浦友亮
  • Organizer: 第8回無細胞生命科学研究会
  • Place of Presentation: 新潟大学
• [Presentation] リポソーム内部合成膜タンパク質はベシクルサイズ依存的に膜挿入される (2013)
  • Author(s): 曽我遥 加藤泰彦 渡邉肇 松浦友亮
  • Organizer: 第8回無細胞生命科学研究会
  • Place of Presentation: 新潟大学
• [Presentation] リポソーム内無細胞膜タンパク質合成系ヘのトランスロコンの導入 (2013)
  • Author(s): 太田直樹 渡邉肇 松浦友亮
  • Organizer: 第8回無細胞生命科学研究会
  • Place of Presentation: 新潟大学
• [Presentation] 無細胞タンパク質合成系を用いた構成的アプローチ (2013)
  • Author(s): 松浦友亮
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸国際会議場
  • Invited
• [Remarks] 渡邉研ホームページ
  • URL: http://www.bio.eng.osaka-u.ac.jp/ez/project/system.html
• [Patent(Industrial Property Rights)] 改良型α-ヘモリシンおよひそれを用いたナノポアシーケンサ (2013)
  • Inventor(s): 四方哲也、松浦友亮、數田恭章、藤井聡志
  • Industrial Property Rights Holder: 四方哲也、松浦友亮、數田恭章、藤井聡志
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2013-157799
  • Filing Date: 2013-10-10