title: Construction of an in Vitro Gene Screening System of the E. coli EmrE Transporter Using Liposome Display.
abstract: Liposome display is a 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 from a single copy of template DNA. So far, a large number of membrane proteins have been synthesized by IVTT; however, none of these proteins, except for α-hemolysin, has been tested for use in gene screening with liposome display. Here, using EmrE, a multidrug transporter from Escherichia coli,, as a model protein, we developed an in vitro screening system of the transporter gene based on its function, which was made possible by using liposome display. 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, which was in the same range as theoretically calculated values. In addition, starting from a random mutagenized gene library of wild-type EmrE, a gene pool exhibiting ethidium bromide (EtBr) transport activity higher than that of the wild-type was obtained, indicating the validity of the established screening system.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Uyeda
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Nakayama
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University , 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Tomoaki
authors.identifier: 0000-0003-1015-6781
authors.initials: T
authors.lastname: Matsuura
mesh_terms: D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008081:Liposomes; C075436:EmrE protein, E coli; D004996:Ethidium