title: Liposome display for in vitro selection and evolution of membrane proteins.
abstract: Liposome display is a novel method for in vitro selection and directed evolution of membrane proteins. In this approach, membrane proteins of interest are displayed on liposome membranes through translation from a single DNA molecule by using an encapsulated cell-free translation system. The liposomes are probed with a fluorescence indicator that senses membrane protein activity and selected using a fluorescence-activated cell sorting (FACS) instrument. Consequently, DNA encoding a protein with a desired function can be obtained. By implementing this protocol, researchers can process a DNA library of 10(7) different mutants. A single round of the selection procedure requires 24 h for completion, and multiple iterations of this technique, which take 1-5 weeks, enable the isolation of a desired gene. As this protocol is conducted entirely in vitro, it enables the engineering of various proteins, including pore-forming proteins, transporters and receptors. As a useful example of the approach, here we detail a procedure for the in vitro evolution of α-hemolysin from Staphylococcus aureus for its pore-forming activity.
authors.affiliation: Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Fujii
authors.affiliation: 1] Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan. [2] Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.affiliation: 1] Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan. [2] Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sunami
authors.affiliation: Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Nishikawa
authors.affiliation: Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kazuta
authors.affiliation: 1] Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan. [2] Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan. [3] Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D002474:Cell-Free System; D019020:Directed Molecular Evolution / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D015723:Gene Library; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D013211:Staphylococcus aureus / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D008081:Liposomes; D008565:Membrane Proteins