Protein-probing molecules that work only inside cells have been developed by US scientists.

Ron Raines, from the University of Wisconsin-Madison, US, and colleagues designed a small molecule containing a fluorophore and used it to label and image a target protein in cells. The team's approach relies on enzymes to activate the fluorophore and fuse it to the protein.

Two enzyme-catalysed steps are needed before Raines's latent fluorophore labels its target protein

Raines's label doesn't fluoresce until it is activated by an esterase which is found only inside cells. This means the system has minimal background fluorescence even when high concentrations of label are used, removing the need to wash the cells before monitoring their fluorescence. Wash steps used to rinse away excess fluorescent label are 'time consuming and tedious,' comments Raines.

Along with the esterase-cleavable fragment, the new fluorescent label also contains a haloalkane functional group. This allows the researchers to tag the fluorophore to the protein target using a system called HaloTag. This system uses a variant of a haloalkane dehalogenase - an enzyme that usually catalyses haloalkane hydrolysis. But the variant dehalogenase cannot complete the hydrolysis and halts once the fluorophore is covalently attached to the enzyme, therefore labelling the system. The team says that by using recombinant DNA technology to attach the haloalkane dehalogenase to other cell proteins it will be possible to study a range of proteins inside live cells. 

Raines's system has several advantages as the fluorophore activation and covalent labelling steps are both catalysed by enzymes - meaning they are highly selective and efficient. Also, the fluorescent label is based on a modular system which allows you to change the fluorophore. Spencer Williams, an expert in chemical biology at the University of Melbourne, Australia, explains that 'the benefit of chemical dyes over fluorescent proteins is the relative ease with which the colour may be tuned.' By tagging a protein with different coloured fluorophores at different times the researchers suggest they could conduct time-dependent studies, which isn't possible using fluorescent proteins. 

Jin Zhang, an expert in fluorescence imaging at the Johns Hopkins University, Baltimore, US, also welcomes the research. Fluorophores are one of the most important factors in the cellular labelling equation, she says. 'This work not only produced a useful latent fluorophore that causes minimal background fluorescence to further advance HaloTag labelling technology, but also investigated the factors that contribute to the desirable properties, paving the way for future development.'  

Russell Johnson

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