Miro-Vinyals, CarlaStein, AlinaFischer, SandroWard, Thomas R.Liang, Alexandria Deliz2021-11-062021-11-062021-11-062021-10-1910.1002/cbic.202100424https://infoscience.epfl.ch/handle/20.500.14299/182824WOS:000708557900001HaloTag 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.Biochemistry & Molecular BiologyChemistry, MedicinalBiochemistry & Molecular BiologyPharmacology & Pharmacydirected evolutionhalotaglive-cell imagingprotein engineeringgeneral-methodlive-cellfluorophorestext::journal::journal article::research article