Selective labelling of target proteins with fluorescent probes in live cells is an important tool for studying protein functions, interactions, and movements in vivo. Various fluorescent proteins has been the probe of choice as they can easily be incorporated to target proteins by recombinant DNA technology. Recently, various approaches are also developed for facile and effective introduction of small synthetic probes of enhanced fluorescent properties onto target proteins.1 Synthetic probes are expected to complement the shortcomings of fluorescent protein (FP) based approaches by widening the colour pallets and allowing modular approaches for multi-colour labelling. Synthetic probes can also be utilized as controllable probes, i.e., photosensitive probes. As controllable probes are powerful tools for the investigation of cellular processes with proper spatiotemporal resolution, there is growing interest in developing fluorescent probes that can be controlled using external stimuli.2 In this study, we designed an approach to photo-chemically trigger fluorescent labelling of target proteins in live cells. Our approach is based on recently developed method to label protein of interest (POI) by using Npu DanE intein-mediated protein trans-splicing reaction (PTS) in live cells.3 Split-inteins fold together to form a functionally active splicing domain allowing the modular approach to incorporate synthetic residues to POI. The developed method has several advantages compared to previously reported photosensitive probes. First, PTS allows rapid linker-free labelling for minimal disruption of endogenous protein functions, second, the use of a FRET-quenched fluorophore enables wash-free, background-free labelling of target proteins, and third, this approach can be generically applied to various fluorophores. The developed method has several advantages compared to previously reported photosensitive probes.

Photo-triggered fluorescent labelling of recombinant proteins in living cells

256th National Meeting (2018)

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