BromoCatch: a self-labelling tag platform for protein analysis and live cell imaging

ABSTRACT: Visualizing and manipulating proteins in live cells is crucial for studying complex biological processes. Self-labelling protein (SLP) tags such as HaloTag and SNAP-tag can be fused to genes of interest to allow protein labelling in cells. New tools to enable rapid, specific and stable protein labelling are in demand to expand the arsenal of SLPs. Here we present BromoCatch, a novel SLP platform based on a small ~13 kDa bromodomain (BD) engineered with a nucleophilic cysteine for covalent ligand engagement. A structure-based designed library of 16 “bumped” binders bearing diverse electrophilic warheads was screened against two cysteine mutants using differential scanning fluorimetry and intact protein mass spectrometry to monitor covalent complex formation. We qualify the para-acrylamide bumped derivative MR116 and the Brd4-BD2 double mutant L387A,E438C as the protein-ligand pair of choice, and its covalent binding mode was confirmed by an X-ray cocrystal structure solved to 1.3 Å of resolution. The BromoCatch platform exhibited potent and irreversible target engagement in cells using nanoBRET and residence time assays. Practicality and scope are further demonstrated through the design and proof-of-concept application of a biotinylated conjugate, PROTAC tag degraders, and fluorescent probes of both full-on and switch-on types for ex-cellulo and live-cell imaging, including side-by-side comparison and orthogonality with HaloTag. Together, we qualify BromoCatch as a novel, versatile and efficient protein labelling tool. Its advantageous design features and kinetic fitness enable modular attachment of diverse functionalities, multiplexing with other SLPs, and catalytic degradation, which will open future applications and witness broad utility.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Early Embryonic Cell

SUBMITTER: Gajanan Sathe  

LAB HEAD: Alessio Ciulli

PROVIDER: PXD074567 | Pride | 2026-05-04

REPOSITORIES: Pride