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

Lin, Y.-H.; Kompa, J.; Sun, D.-e.; Mao, R.; Koch, B.; Hinnah, K.; Wilhelm, J.; Franz, N.; Kühn, S.; Menche, T.; Adow, A.; Breuer, P.; Hiblot, J.; Johnsson, K.

doi:10.1101/2025.06.15.659771

bioRxiv · 10.1101/2025.06.15.659771

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

Lin, Y.-H.·Kompa, J.·Sun, D.-e.·Mao, R.·Koch, B.·Hinnah, K.·Wilhelm, J.·Franz, N.·Kühn, S.·Menche, T.·Adow, A.·Breuer, P.·Hiblot, J.·Johnsson, K.

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Abstract

We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHalo{Delta}3). Hpep binds to cpHalo{Delta}3 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 cpHalo{Delta}3 and in fixed cells through incubation with cpHalo{Delta}3. 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.

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Lin, Y.-H., Kompa, J., Sun, D.-e., Mao, R., Koch, B., Hinnah, K., Wilhelm, J., Franz, N., Kühn, S., Menche, T., Adow, A., Breuer, P., Hiblot, J., Johnsson, K.. 2025-06-15. A high-affinity split-HaloTag for live-cell protein labeling. https://doi.org/10.1101/2025.06.15.659771

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