Liposome display for in vitro selection and evolution of membrane proteins

被引：124

作者：

Fujii, Satoshi ^{[1
]}

Matsuura, Tomoaki ^{[1
,2
]}

Sunami, Takeshi ^{[1
,3
]}

Nishikawa, Takehiro ^{[1
]}

Kazuta, Yasuaki ^{[1
]}

Yomo, Tetsuya ^{[1
,3
,4
]}

机构：

[1] Japan Sci & Technol Agcy, Exploratory Res Adv Technol ERATO, Yomo Dynam Microscale React Environm Project, Osaka, Japan

[2] Osaka Univ, Sch Engn, Dept Biotechnol, Osaka, Japan

[3] Osaka Univ, Grad Sch Informat Sci & Technol, Dept Bioinformat Engn, Osaka, Japan

[4] Osaka Univ, Grad Sch Frontier Biosci, Osaka, Japan

来源：

NATURE PROTOCOLS | 2014年 / 9卷 / 07期

关键词：

STAPHYLOCOCCAL ALPHA-HEMOLYSIN; LIBRARIES; SYSTEM; BACTERIORHODOPSIN; INSERTION; PEPTIDES; SYNTHASE;

D O I：

10.1038/nprot.2014.107

中图分类号：

Q5 [生物化学];

学科分类号：

070307 [化学生物学];

摘要：

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 107 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 alpha-hemolysin from Staphylococcus aureus for its pore-forming activity.

引用

页码：1578 / 1591

页数：14

共 44 条

[21]

In vitro virus: Bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro [J].