| Budget Amount *help |
¥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)
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| 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.
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