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Fluorogen-activating single-chain antibodies for imaging cell surface proteins

A Corrigendum to this article was published on 01 April 2008

This article has been updated

Abstract

Imaging of live cells has been revolutionized by genetically encoded fluorescent probes, most famously green and other fluorescent proteins, but also peptide tags that bind exogenous fluorophores1. We report here the development of protein reporters that generate fluorescence from otherwise dark molecules (fluorogens). Eight unique fluorogen activating proteins (FAPs) have been isolated by screening a library of human single-chain antibodies (scFvs) using derivatives of thiazole orange and malachite green. When displayed on yeast or mammalian cell surfaces, these FAPs bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. Spectral variation can be generated by combining different FAPs and fluorogen derivatives. Visualization of FAPs on the cell surface or within the secretory apparatus of mammalian cells can be achieved by choosing membrane permeant or impermeant fluorogens. The FAP technique is extensible to a wide variety of nonfluorescent dyes.

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Figure 1: Fluorogens and their use with yeast-displayed scFvs.
Figure 2: Fluorescence characterization of purified FAPs.
Figure 3: Fluorescence microscopy of FAPs displayed on live cells.

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Change history

  • 19 March 2008

    In the version of this article initially published online, an author’s middle initial was incorrectly given as “A” Brigitte A Schmidt should be Brigitte F Schmidt in the author list, and B.A.S should be B.F.S. in the authors’ contribution section. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank Dane Wittrup for providing an aliquot of the yeast scFv surface display library, members of his laboratory for advice and Nicholas Bateman for work on scFv maturation. Supported by National Institutes of Health grant 7 U54 RR022241 and Pennsylvania Department of Health grant 4100020575.

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Contributions

B.F.S. synthesized fluorogens and derivatives; C.S.-G. and Y.C. cloned fluorogenic scFvs under the direction of C.S.-G.; K.L.Z. carried out cloning-related molecular biology and protein purification; under the direction of J.W.J., S.A. constructed mammalian expression vectors with contribution from K.V.V.; G.W.F. and Q.Y. created stable mammalian cell lines; G.W.F. and J.A.J.F. performed microscopy; C.A.W. implemented directed evolution mutagenesis with contribution from P.B.B.; C.S.-G. conducted spectroscopy and binding experiments and analysis; C.S.-G. drafted manuscript and figures; M.P.B., J.W.J., P.B.B. and A.W. contributed editorially to manuscript and to general design of research; A.W. conceived fluorogen strategy and oversaw entire project.

Corresponding authors

Correspondence to Christopher Szent-Gyorgyi or Alan Waggoner.

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Supplementary Tables 1, Figures 1–8, Methods (PDF 1556 kb)

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Szent-Gyorgyi, C., Schmidt, B., Creeger, Y. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat Biotechnol 26, 235–240 (2008). https://doi.org/10.1038/nbt1368

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