Expanding the HaloTag toolbox: A new tool to fish out protein variants from live cells

Proteins inside our cells are incredibly dynamic. They exist in different forms, change locations, and take on distinct roles depending on what the cell needs. To study these differences, scientists use technologies that help them ‚tag‘ proteins so they can be seen under a microscope or pulled out for closer examination. One of the most widely used systems for this is the HaloTag technology, which allows researchers to label proteins with fluorescent dyes and track their behaviour inside living cells.

In recent years, HaloTag-based imaging has revealed that many essential proteins, like histones, cohesins, or MCMs (involved in DNA replication), can exist in multiple variants with different functions. But while imaging tells part of the story, scientists have lacked a good way to biochemically separate and study these different protein forms. The missing piece has been a reliable tool that works inside live cells to isolate tagged proteins based on their age and thus their function.

In a collaborative effort between the Institute of Biophysics of the Czech Academy of Sciences, Masaryk University, and CEITEC, researchers have now developed a new biotin-HaloTag ligand—a small molecule that can easily enter live cells, label Halo-tagged proteins, and allow them to be pulled out using standard streptavidin-based techniques. This method works quickly and efficiently, making it possible to capture and analyze distinct protein variants based on their age from live cells.

A graphical summary describing the new biotin-HaloTag ligand tool and its application.

Anoop Kumar Yadav (PhD student in Sedlackova laboratory), Dr. Abhijeet S. Jadhav (postdoc from Svenda laboratory), and their colleagues demonstrated how this new tool can be used in sequential labeling experiments to biochemically isolate different versions of the same protein. This opens the door to studying how protein variants contribute to vital processes like DNA replication and chromatin organization, especially under changing cellular conditions.

By combining live-cell labeling with Biochemical methods, this work provides a valuable tool for understanding how cells maintain their genome and respond to stress, a foundation for exploring diseases like cancer at the molecular level.

You can read the full article here.

This research was supported by the Czech Science Foundation (22-20303M), European Union (ERA grant agreement no. 101090292), the EMBO Installation Grant (IG-5689-2024), and funding from collaborating institutions.