Publication

A high-affinity split-HaloTag for live-cell protein labeling

Kai Johnsson
2025

Abstract

We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHaloΔ3). Hpep binds to cpHaloΔ3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHaloΔ3 and in fixed cells through incubation with cpHaloΔ3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.

Official source

https://infoscience.epfl.ch/entities/publication/2c2b66bb-629d-4ca3-94e9-1c631cc6c704

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Kai Johnsson
2025

Abstract

Official source

https://infoscience.epfl.ch/entities/publication/2c2b66bb-629d-4ca3-94e9-1c631cc6c704

About this result

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A high-affinity split-HaloTag for live-cell protein labeling

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