title: Fluorogen-activating single-chain antibodies for imaging cell surface proteins.
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.
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. css@andrew.cmu.edu
authors.forename: Christopher
authors.initials: C
authors.lastname: Szent-Gyorgyi
authors.forename: Brigitte F
authors.initials: BF
authors.lastname: Schmidt
authors.forename: Brigitte A
authors.initials: BA
authors.lastname: Schmidt
authors.forename: Yehuda
authors.initials: Y
authors.lastname: Creeger
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Fisher
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Zakel
authors.forename: Sally
authors.initials: S
authors.lastname: Adler
authors.forename: James A J
authors.initials: JA
authors.lastname: Fitzpatrick
authors.forename: Carol A
authors.initials: CA
authors.lastname: Woolford
authors.forename: Qi
authors.initials: Q
authors.lastname: Yan
authors.forename: Kalin V
authors.initials: KV
authors.lastname: Vasilev
authors.forename: Peter B
authors.initials: PB
authors.lastname: Berget
authors.forename: Marcel P
authors.initials: MP
authors.lastname: Bruchez
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Jarvik
authors.forename: Alan
authors.initials: A
authors.lastname: Waggoner
mesh_terms: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins