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

Angew Chem Int Ed Engl. 2022 Sep 5;61(36):e202207905. doi: 10.1002/anie.202207905. Epub 2022 Jul 29.

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.

Keywords: Covalent Drug; Live-Cell Imaging; Orthogonal Bioconjugation; Protein Loop Engineering; Self-Labeling Tag.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli*
  • Fluorescent Dyes
  • Proteins
  • Rhodamines
  • Tetrahydrofolate Dehydrogenase
  • Trimethoprim*

Substances

  • Fluorescent Dyes
  • Proteins
  • Rhodamines
  • Trimethoprim
  • Tetrahydrofolate Dehydrogenase