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<p>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 are widely used for protein labelling, and new systems are needed to expand multiplexing capabilities and broaden the scope of applications. Here we present BromoCatch, a small ~13 kDa bromodomain&#xa0;(BD)-based SLP platform, engineered with a nucleophilic cysteine for covalent ligand engagement. A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation. We identified a para-acrylamide bumped derivative MR116 and the Brd4-BD2 double mutant L387A,E438C as the optimal protein-ligand pair, and reveal the binding mode through an X-ray co-crystal structure solved to 1.3 Å&#xa0;resolution. BromoCatch demonstrated potent and irreversible cellular target engagement in NanoBRET and residence-time assays. Its versatility was demonstrated through the design of a biotinylated conjugate, PROTAC-based degraders, and fluorescent full-on and “switch-on” probes for ex-cellulo and live-cell imaging, including side-by-side comparison and orthogonality with HaloTag. Together, these results establish BromoCatch as a robust, modular, and orthogonal SLP tool with broad potential for multiplexed labelling and targeted protein manipulation.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月13日

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  • Maria Rodriguez-Rios,
  • Conner Craigon,
  • Mark A. Nakasone,
  • Gajanan Sathe,
  • Adam G. Bond,
  • Mark Dorward,
  • Anthony K. Edmonds,
  • Mark C. Norley,
  • Robert E. Arnold,
  • Paul M. Wood,
  • Stephen J. Reynolds,
  • Joel O. Cresser-Brown,
  • Graham P. Marsh,
  • Hannah J. Maple,
  • Alessio Ciulli

摘要

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 are widely used for protein labelling, and new systems are needed to expand multiplexing capabilities and broaden the scope of applications. Here we present BromoCatch, a small ~13 kDa bromodomain (BD)-based SLP platform, engineered with a nucleophilic cysteine for covalent ligand engagement. A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation. We identified a para-acrylamide bumped derivative MR116 and the Brd4-BD2 double mutant L387A,E438C as the optimal protein-ligand pair, and reveal the binding mode through an X-ray co-crystal structure solved to 1.3 Å resolution. BromoCatch demonstrated potent and irreversible cellular target engagement in NanoBRET and residence-time assays. Its versatility was demonstrated through the design of a biotinylated conjugate, PROTAC-based degraders, and fluorescent full-on and “switch-on” probes for ex-cellulo and live-cell imaging, including side-by-side comparison and orthogonality with HaloTag. Together, these results establish BromoCatch as a robust, modular, and orthogonal SLP tool with broad potential for multiplexed labelling and targeted protein manipulation.