Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.
Representative scheme of Haloalkane_dehalogenase-HLD2 structure and an image from PDBsum server
Databases
PDB-Sum
7PCW Previously Class, Architecture, Topology and Homologous superfamily - PDB-Sum server
FSSP
7PCWFold classification based on Structure-Structure alignment of Proteins - FSSP server
