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

## TL;DR

A new split-HaloTag system enables efficient and flexible live-cell protein labeling with high affinity and compatibility with advanced imaging techniques.

## Contribution

The development of a high-affinity split-HaloTag system with a short peptide tag and a large inactive fragment for live-cell protein labeling.

## Key findings

- The split-HaloTag system enables spontaneous binding with nanomolar affinity for fluorescent labeling.
- The system supports endogenous tagging without cloning and works in both live and fixed cells.
- Variants of the peptide tag allow multiplexed imaging of proteins using fluorescence lifetime microscopy.

## 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.

Lin and colleagues present high-affinity split-HaloTag pairs for protein tagging and multiplexed labelling. This versatile system allows protein visualisation with diverse imaging modalities, including live-cell super-resolution microscopy.