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Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications

Nature Protocols volume 9, pages 910–928 (2014)Cite this article

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Abstract

Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4–6 weeks.

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Figure 1: Steps used to engineer a new FP.
Figure 2: Conversion of a naturally tetrameric FP to an engineered monomeric FP.
Figure 3: Typical photobleaching kinetics of FPs.
Figure 4
Figure 5: Mounting procedure for photobleaching proteins.
Figure 6: Comparison of good and poor localization of FP fusions in similar constructs.

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Acknowledgements

This research was made possible with financial support from the University of Alberta, the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council of Canada, and Alberta Ingenuity (scholarship to Y.S. and a New Faculty Award to R.E.C.). R.E.C. holds a Canada Research Chair in Bioanalytical Chemistry.

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Authors and Affiliations

  1. Department of Chemistry, University of California, Riverside, California, USA

    Hui-wang Ai

  2. National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida, USA

    Michelle A Baird & Michael W Davidson

  3. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Yi Shen & Robert E Campbell

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  1. Hui-wang Ai
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Contributions

M.W.D. and M.A.B. acquired data and images shown in Figures 3 and 6. H.A., M.A.B., Y.S., M.W.D. and R.E.C. developed the protocols and wrote and edited the paper.

Corresponding authors

Correspondence to Michael W Davidson or Robert E Campbell.

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Ai, Hw., Baird, M., Shen, Y. et al. Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications. Nat Protoc 9, 910–928 (2014). https://doi.org/10.1038/nprot.2014.054

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