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

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 mole...

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Bibliographic Details
Published in:Nature biotechnology 2008-02, Vol.26 (2), p.235-240
Main Authors: Szent-Gyorgyi, Christopher, Waggoner, Alan, Schmidt, Brigitte F, Creeger, Yehuda, Fisher, Gregory W, Zakel, Kelly L, Adler, Sally, Fitzpatrick, James A J, Woolford, Carol A, Yan, Qi, Vasilev, Kalin V, Berget, Peter B, Bruchez, Marcel P, Jarvik, Jonathan W
Format: Article
Language:English
Subjects:
Agriculture
Antibodies
Antibodies, Monoclonal
Bioinformatics
Biological and medical sciences
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Cellular biology
Cellular proteins
Dyes
Fluorescence
Fluorescent Dyes
Fundamental and applied biological sciences. Psychology
Genes, Reporter
Genetic aspects
Green fluorescent protein
Health aspects
Immunoglobulin Fragments
letter
Life Sciences
Mammals
Membrane Proteins - metabolism
Membrane Proteins - ultrastructure
Microscopy, Fluorescence - methods
Molecular Probe Techniques
Physiological aspects
Probes
Proteins
Scientific imaging
Viral antibodies
Yeasts
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Summary: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.
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt1368