Third‐Generation Covalent TMP‐Tag for Fast Labeling and Multiplexed Imaging of Cellular Proteins

摘要

Abstract Self‐labeling protein tags can introduce advanced molecular motifs to specific cellular proteins. Here we introduce the third‐generation covalent TMP‐tag (TMP‐tag3) and showcase its comparison with HaloTag and SNAP‐tag. TMP‐tag3 is based on a proximity‐induced covalent Michael addition between an engineered Cys of E. coli dihydrofolate reductase (eDHFR) and optimized trimethoprim (TMP)–acrylamide conjugates with minimal linkers. Compared to previous versions, the TMP‐tag3 features an enhanced permeability when conjugated to fluorogenic spirocyclic rhodamines. As a small protein, the 18‐kD eDHFR is advantageous in tagging selected mitochondrial proteins which are less compatible with bulkier HaloTag fusions. The proximal N?C termini of eDHFR also enable facile insertion into various protein loops. TMP‐tag3, HaloTag, and SNAP‐tag are orthogonal to each other, collectively forming a toolbox for multiplexed live‐cell imaging of cellular proteins under fluorescence nanoscopy.