Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

In addition, these FAPs contained internal disulfide bonds, which restricted their use to non-reducing environments such as the cell surface and secretory apparatus, since these FAPs may not fold properly in the reducing environment of the cytosol. The engineering of disulfide-free FAPs, like p13-CW FAP, a classic heavy-light scFv (HL4) with the second cysteine in each domain changed to an alanine, improved labeling in the cytoplasm and various other reducing subcellular compartments 24, 25 . Furthermore, selection of scFvs against other fluorogens successfully extended the chromatic palette of FAPs 26, 27 . Of particular interest, some scFv promiscuously activate various dimethylindole red (DIR) analogs, providing access to wavelengths ranging from the blue to the near infrared (NIR, 650-900 nm) 26 . There are also promiscuous FAPs that can bind more than one fluorogen, with alternate excitation and emission wavelengths and varying affinity constants for fluorogen binding 21 . Further improvements in brightness would result in better sensitivity and lower phototoxicity under typical imaging conditions.