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 α-hemolysin from Staphylococcus aureus for its pore-forming activity.
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We thank H. Komai, T. Sakamoto and R. Otsuki for their technical assistance. This research was supported in part by the Global Centers of Excellence Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Histograms representing the size distribution of liposome during the procedure of liposome display.
Liposome was constructed and treated as described in steps 4–22. The horizontal axis shows the size of the unilamellar liposome and vertical axis shows the number of unilamellar liposome measured by FACS in 100 s. The liposome suspensions obtained after liposome construction (step 15), centrifugation (step 17), 4-h incubation (step 18), and ligand addition (step 22) were analyzed. The condition of FACS measurement was set as described in EQUIPMENT SETUP. Significant loss of liposome was not observed.
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Fujii, S., Matsuura, T., Sunami, T. et al. Liposome display for in vitro selection and evolution of membrane proteins. Nat Protoc 9, 1578–1591 (2014). https://doi.org/10.1038/nprot.2014.107
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