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Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library

Nature Biotechnology volume 24, pages 1569–1571 (2006)Cite this article

Abstract

The utility of blue fluorescent protein (BFP) has been limited by its low quantum yield and rapid photobleaching. A library targeting residues neighboring the chromophore yielded a variant with enhanced quantum yield (0.55 versus 0.34), reduced pH sensitivity and a 40-fold increase in photobleaching half-life. This BFP, named Azurite, is well expressed in bacterial and mammalian cells and extends the palette of fluorescent proteins that can be used for imaging.

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Figure 1: Screening and characterization of BFP variants.

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Acknowledgements

The authors thank K. Dane, A. Nguyen, P. Bessette and C. Gottstein for helpful discussions and Alexander Mikhailovsky for assistance with fluorescence lifetime measurements, and acknowledge support from National Institutes of Health–National Institute of Biomedical Imaging and Bioengineering grant EB-000205.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, University of California, Santa Barbara, 93106, California, USA

    Marco A Mena & Patrick S Daugherty

  2. Institute for Collaborative Biotechnologies, University of California, Santa Barbara, 93106, California, USA

    Stephen L Mayo & Patrick S Daugherty

  3. Biomolecular Science and Engineering, University of California, Santa Barbara, 93106, California, USA

    Patrick S Daugherty

  4. Division of Biology, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Thomas P Treynor & Stephen L Mayo

Authors
  1. Marco A Mena
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  2. Thomas P Treynor
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  3. Stephen L Mayo
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  4. Patrick S Daugherty
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Contributions

M.A.M., T.P.T., S.L.M. and P.S.D. designed research, M.A.M. and T.P.T. performed research, T.P.T. and S.L.M. contributed computational tools, M.A.M., T.P.T. and P.S.D. analyzed data, and M.A.M. and P.S.D. wrote the paper.

Corresponding author

Correspondence to Patrick S Daugherty.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Crystal structure of BFP with chromophore (blue) and targeted residues (red). (PDF 225 kb)

Supplementary Fig. 2

Photophysical characterization of BFP variants. (PDF 18 kb)

Supplementary Fig. 3

Fluorescence lifetime analysis of BFP mutants. (PDF 25 kb)

Supplementary Fig. 4

Fluorescence of BFP variants with varying pH. (PDF 14 kb)

Supplementary Fig. 5

Whole-cell fluorescence development of E. coli cells expressing Azurite and EGFP. (PDF 18 kb)

Supplementary Fig. 6

Non-denaturing polyacrylamide gel electrophoresis of BFP variants. (PDF 742 kb)

Supplementary Fig. 7

Alignment of wtGFP, BFP, A5, and Azurite Amino Acid Sequences. (PDF 31 kb)

Supplementary Fig. 8

Alignment of WT GFP, BFP, A5 and Azurite Amino Acid sequences. (PDF 30 kb)

Supplementary Table 1

Amino acid composition of BFP degenerate codon library (PDF 14 kb)

Supplementary Table 2

Degenerate codon library design for structurally targeted mutagenesis and isolated BFP variants (PDF 15 kb)

Supplementary Methods (PDF 21 kb)

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Mena, M., Treynor, T., Mayo, S. et al. Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library. Nat Biotechnol 24, 1569–1571 (2006). https://doi.org/10.1038/nbt1264

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  • DOI: https://doi.org/10.1038/nbt1264

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