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Liposome display for in vitro selection and evolution of membrane proteins

Nature Protocols volume 9, pages 1578–1591 (2014)Cite this article

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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 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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Figure 1: Overview of the stages involved in liposome construction and screening of DNA when implementing the liposome display method.
Figure 2: Time course of AF488 ligand accumulation with or without the addition of a biotinylated ligand after 60 min of incubation (dashed line).
Figure 3: Typical 2D plots and a histogram of liposomes obtained through FACS.
Figure 4: Stages of liposome construction using the water-in-oil emulsion transfer method.
Figure 5: Functional analysis and sequence identification of α-hemolysin mutants obtained after 20 rounds of selection.

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Acknowledgements

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.

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Authors and Affiliations

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

    Satoshi Fujii, Tomoaki Matsuura, Takeshi Sunami, Takehiro Nishikawa, Yasuaki Kazuta & Tetsuya Yomo

  2. Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan

    Tomoaki Matsuura

  3. Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, Osaka, Japan

    Takeshi Sunami & Tetsuya Yomo

  4. Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

    Tetsuya Yomo

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Contributions

S.F., T.M. and T.Y. designed the research; S.F. performed experiments; S.F., T.S. and T.N. developed the methods; Y.K. contributed reagents; T.S. and T.N. commented on the paper; and S.F. and T.M. wrote the manuscript.

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Correspondence to Tetsuya Yomo.

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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.

Supplementary information

Supplementary Figure 1

Histograms representing the size distribution of liposome during the procedure of liposome display. (PDF 89 kb)

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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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