A high-affinity split-HaloTag for live-cell protein labeling
- Yin-Hsi Lin
- Julian Kompa
- De-en Sun
- Runyu Mao
- Birgit Koch
- Konstantin Hinnah
- Jonas Wilhelm
- Natascha Franz
- Stefanie Kühn
- Tanja Menche
- Abdinasir Adow
- Paula Breuer
- Julien Hiblot
- Kai Johnsson
2026-03-25
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.