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

2015 Fiscal Year Final Research Report

• 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
• Keywords: 進化分子工学 / 無細胞翻訳系

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.

Free Research Field

生物工学