HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye

HaloTag is a small self‐labeling protein that is frequently used for creating fluorescent reporters in living cells. The small‐molecule dyes used with HaloTag are almost exclusively based on rhodamine scaffolds, which are often expensive and challenging to synthesize. Herein, we report the engineering of HaloTag for use with a chemically accessible, inexpensive fluorophore based on the dimethylamino‐styrylpyridium dye. Through directed evolution, the maximum fluorogenicity and the apparent second‐order bioconjugation rate constants could be improved up to 4‐fold and 42‐fold, respectively. One of the top variants, HT‐SP5, enabled reliable imaging in mammalian cells, with a 113‐fold fluorescence enhancement over the parent protein. Additionally, crystallographic characterization of selected mutants suggests the chemical origin of the fluorescent enhancement. The improved dye system offers a valuable tool for imaging and illustrates the viability of engineering self‐labeling proteins for alternative fluorophores.