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Publication Abstract from PubMed

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.

HaloTag Engineering for Enhanced Fluorogenicity and Kinetics with a Styrylpyridium Dye.,Miro-Vinyals C, Stein A, Fischer S, Ward TR, Deliz Liang A Chembiochem. 2021 Oct 5. doi: 10.1002/cbic.202100424. PMID:34609782^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.