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Reporter proteins for in vivo fluorescence without oxygen

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Abstract

Fluorescent reporter proteins such as green fluorescent protein are valuable noninvasive molecular tools for in vivo real-time imaging of living specimens. However, their use is generally restricted to aerobic systems, as the formation of their chromophores strictly requires oxygen. Starting with blue-light photoreceptors from Bacillus subtilis and Pseudomonas putida that contain light-oxygen-voltage–sensing domains, we engineered flavin mononucleotide–based fluorescent proteins that can be used as fluorescent reporters in both aerobic and anaerobic biological systems.

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Figure 1: Generation, characterization and in vivo application of anaerobically fluorescing proteins.

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Acknowledgements

The authors would like to thank Jürgen Hubbuch and Thomas Rosenbaum, Institute of Biotechnology 2, Forschungszentrum Jülich, Germany, for their assistance with confocal laser scanning microscopy. This work was partly supported by grants from the Deutsche Forschungsgemeinschaft (DFG) within the Forschergruppe FOR526 “Sensory blue light photoreceptors.”

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Author notes

  1. Thomas Drepper and Thorsten Eggert: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular Enzyme Technology, Heinrich-Heine-University Duesseldorf, Research Center Juelich, Stetternicher Forst, Juelich, D-52426, Germany

    Thomas Drepper, Thorsten Eggert, Franco Circolone, Achim Heck, Ulrich Krauß, Jan-Karl Guterl, Marion Wendorff & Karl-Erich Jaeger

  2. EVOcatal GmbH, Merowingerplatz 1a, Duesseldorf, D-40225, Germany

    Thorsten Eggert

  3. Department of Physics, University of Parma, viale G.P. Usberti 7/A, Parma, I-43100, Italy

    Aba Losi

  4. Max-Planck-Institute for Bioinorganic Chemistry, Stiftstraße 34-36, Mülheim an der Ruhr, D-45470, Germany

    Wolfgang Gärtner

Authors
  1. Thomas Drepper
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  2. Thorsten Eggert
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  3. Franco Circolone
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  4. Achim Heck
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  5. Ulrich Krauß
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  6. Jan-Karl Guterl
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  7. Marion Wendorff
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  8. Aba Losi
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  9. Wolfgang Gärtner
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  10. Karl-Erich Jaeger
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Corresponding authors

Correspondence to Thomas Drepper or Karl-Erich Jaeger.

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Competing interests

A patent application has been filed covering part of the results described in this manuscript (T.E., T.D., J.-K.G., A.H. & K.-E.J., patent no. DE 10 2005 048 828.5 (2005)).

Supplementary information

Supplementary Fig. 1

Structure of the Chlamydomonas reinhardtii LOV1 domain (pdb entry: 1N9L) in the dark state. (DOC 584 kb)

Supplementary Fig. 2

The complete nucleotide and amino acid sequences of BsFbFP, EcFbFP, and PpFbFP. (DOC 30 kb)

Supplementary Methods

(DOC 47 kb)

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Drepper, T., Eggert, T., Circolone, F. et al. Reporter proteins for in vivo fluorescence without oxygen. Nat Biotechnol 25, 443–445 (2007). https://doi.org/10.1038/nbt1293

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