Angewandte Chemie · 2015 · 11 citations · 18 references
Life SciencesBiochemistryMolecular BiologyMolecular ImagingProtein EngineeringStructural BiologyFluorescence MicroscopyMacromolecular Structure DeterminationProtein DesignProtein DimerizationProtein StructureSingle-molecule Fluorescence MicroscopyFluorescence Labeling StrategiesFluorescent MaterialsFluorescent Reporter DesignGenetically Encoded Fluorescent ReportersImmunofluorescence AssaysImmunofluorescence StainingPh SensingProtein Fluorescence
Abstract Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the “vGFP strategy” based on structural analysis of GFP bound to a single‐domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50 %), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single‐molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single‐step immunofluorescence in vitro (3× brighter due to dimerization); expression in bacteria and human cells in vivo (1.5× brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general.
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Engineering and characterization of a superfolder green fluorescent protein
J.D. Pédelacq, Stéphanie Cabantous, Timothy H. Tran et al. · Nature Biotechnology · 2005 · 2.5K citations