Liposome display for in vitro selection and evolution of membrane proteins

• Published in: Nature protocols Vol. 9; no. 7; pp. 1578 - 1591
• Main Authors: Fujii, Satoshi, Matsuura, Tomoaki, Sunami, Takeshi, Nishikawa, Takehiro, Kazuta, Yasuaki, Yomo, Tetsuya
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.07.2014
• Subjects: Analysis; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Cell growth; Cell-Free System; Deoxyribonucleic acid; Directed evolution; Directed Molecular Evolution - methods; DNA; Evolution; Flow cytometry; Flow Cytometry - methods; Fluorescence; Fluorescent indicators; Gene Library; Genetic translation; Genotype & phenotype; Hemolysin Proteins - biosynthesis; Hemolysin Proteins - chemistry; Hemolysin Proteins - genetics; In vitro methods and tests; Lipids; Liposomes; Liposomes - metabolism; Medical research; Membrane proteins; Membrane Proteins - biosynthesis; Membranes; Methods; Pore formation; Pore-forming proteins; Protein Biosynthesis; Protein synthesis; Protein transport; Proteins; Protocol; Ribonucleic acid; RNA; Staphylococcus aureus - genetics; Translation; Japan
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2014.107

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.