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Directed molecular evolution to design advanced red fluorescent proteins

Nature Methods volume 8, pages 1019–1026 (2011)Cite this article

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Abstract

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

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Figure 1: Steps in the directed molecular evolution of fluorescent probes.
Figure 2: Major chemical transformations of the chromophores in red fluorescent proteins.
Figure 3: Methods that could improve molecular evolution of fluorescent proteins.
Figure 4: Possible FACS-based screening approaches for red-shifted fluorescent proteins.

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Acknowledgements

This work was supported by US National Institutes of Health grants GM073913 and CA164468 to V.V.V.

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Author notes

  1. Fedor V Subach and Kiryl D Piatkevich: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Fedor V Subach, Kiryl D Piatkevich & Vladislav V Verkhusha

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  1. Fedor V Subach
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Correspondence to Vladislav V Verkhusha.

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Subach, F., Piatkevich, K. & Verkhusha, V. Directed molecular evolution to design advanced red fluorescent proteins. Nat Methods 8, 1019–1026 (2011). https://doi.org/10.1038/nmeth.1776

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