Format

Send to

Choose Destination
See comment in PubMed Commons below
Nat Protoc. 2014 Jul;9(7):1578-91. doi: 10.1038/nprot.2014.107. Epub 2014 Jun 5.

Liposome display for in vitro selection and evolution of membrane proteins.

Author information

1
Yomo Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency, Osaka, Japan.
2
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.
3
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.
4
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.

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.

PMID:
24901741
DOI:
10.1038/nprot.2014.107
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Nature Publishing Group
    Loading ...
    Support Center