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Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Published on: January 5, 2024

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

Yin-Hsi Lin^1,2, Julian Kompa¹, De-En Sun¹

¹Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.

Nature Communications

|March 25, 2026

Summary

This summary is machine-generated.

Researchers developed a high-affinity split-HaloTag system using a peptide tag (Hpep) and a protein fragment (cpHaloΔ3). This tool enables efficient, cloning-free protein tagging for advanced live-cell imaging and chemical biology applications.

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Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Published on: July 12, 2014

Area of Science:

Chemical Biology
Molecular Imaging
Biotechnology

Background:

Traditional protein tagging methods can be limited by tag size and labeling efficiency.
The HaloTag system offers a robust platform for protein labeling but requires a complete tag for optimal function.

Purpose of the Study:

To develop a high-affinity split-HaloTag system for versatile protein labeling and live-cell imaging.
To create a small peptide tag (Hpep) that enables cloning-free, CRISPR/Cas9-mediated endogenous protein tagging.

Main Methods:

Engineered a split-HaloTag system consisting of a 14-residue peptide tag (Hpep) and a large inactive fragment (cpHaloΔ3).
Demonstrated spontaneous, nanomolar-affinity binding between Hpep and cpHaloΔ3 for subsequent fluorescent ligand labeling.
Utilized CRISPR/Cas9 for cloning-free, endogenous Hpep-tagging of proteins.
Validated complementation in live and fixed cells via co-expression or incubation with cpHaloΔ3.

Main Results:

Achieved high-affinity Hpep-cpHaloΔ3 complementation, enabling efficient protein labeling.
Successfully applied the system for cloning-free, endogenous protein tagging in mammalian cells.
Demonstrated compatibility with advanced microscopy techniques, including expansion microscopy and live-cell STED imaging.
Showcased simultaneous dual-protein imaging using spectral variants of Hpep with fluorescence lifetime microscopy.

Conclusions:

The high-affinity split-HaloTag (Hpep/cpHaloΔ3) is a versatile and robust tool for chemical biology.
This system facilitates advanced live-cell imaging and endogenous protein analysis.
The small Hpep tag enables efficient, non-invasive protein labeling strategies.