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

Christopher Szent-Gyorgyi, Brigitte A. Schmidt, Yehuda Creeger, Gregory W. Fisher, Kelly L. Zakel, Sally Adler, James A.J. Fitzpatrick, Carol A. Woolford, Qi Yan, Kalin V. Vasilev, Peter B. Berget, Marcel P. Bruchez, Jonathan W. Jarvik, Alan Waggoner

Research output: Contribution to journalArticle

248 Scopus citations

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 fluorophores. 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.

Original languageEnglish
Pages (from-to)235-240
Number of pages6
JournalNature Biotechnology
Volume26
Issue number2
DOIs
StatePublished - Feb 1 2008
Externally publishedYes

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    Szent-Gyorgyi, C., Schmidt, B. A., Creeger, Y., Fisher, G. W., Zakel, K. L., Adler, S., Fitzpatrick, J. A. J., Woolford, C. A., Yan, Q., Vasilev, K. V., Berget, P. B., Bruchez, M. P., Jarvik, J. W., & Waggoner, A. (2008). Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nature Biotechnology, 26(2), 235-240. https://doi.org/10.1038/nbt1368