Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

As summarized in this review, fluorogenic labeling is a general concept for imaging biomolecules with high contrast in living systems, with great potential for pushing the limit of biological imaging. In this review, we have discussed in detail about the discovery, development and application of the FAP technology as a novel effective fluorogenic labeling method. Clearly, the FAP- fluorogen system displays a few unprecedented attributes, such as a small size, no-wash, high signal-to-noise ratio, and tunable spectral properties, which make them interesting alternatives to classical fluorescent proteins and open great prospects for advanced imaging, such as super-resolution microscopies. In this two-component system, both the FAP and the fluorogen can be tuned in order to obtain the desired properties. For example, fluorogens can be designed to display improved spectral properties, brightness, and photostability. Meanwhile, through random mutagenesis and directed evolution, FAPs can be selected to accommodate a large repertoire of fluorogens. With further efforts, we expect that the FAP-fluorogen system will be useful for addressing exciting unexplored biological questions and will greatly advance our understanding of human disease and normal cell function. However, in all cases introduced above, the FAP was expressed from a recombinant gene that encoded a protein fusion between the FAP and the protein of interest. This approach results in two significant setbacks 61 : (1) time and labor regarding quality control and generation of each recombinant protein, and (2) artificial protein expression from a nonnative promoter, typically altering protein regulation and abundance in the cell. Therefore, careful upstream studies of their toxicity and their influence on cellular processes is still required. At present, our group is conducting an in-depth study on this issue. Moreover, whether the FAP approach is feasible with smaller antibody fragments such as nanobodies remains to be investigated.