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Updated: Aug 5, 2026
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Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology
Published on: July 12, 2014
HaloTag-Based FACS-Compatible Assay for Intracellular Haloalkane Detection with a Potential Application to Halogenase
Hannes Meinert1, Hannes Wolfgramm2, Hannah Meier1
1Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany.
Analytical Chemistry
|July 27, 2026
Summary
This study introduces a novel flow cytometry assay to detect haloalkanes within cells. This method uses engineered proteins to screen for new halogenase enzymes, advancing drug discovery and chemical synthesis.
Area of Science:
- Biocatalysis
- Synthetic Chemistry
- Molecular Biology
Background:
- Halogenases are crucial for asymmetric Csp3-halogenation, offering control over drug pharmacokinetics.
- Current methods for discovering new halogenases are limited by the lack of high-throughput screening assays.
- Expanding the halogenase toolbox is essential for novel enzymatic halogenation reactions.
Purpose of the Study:
- To develop a high-throughput screening assay for discovering novel halogenases.
- To enable the detection of intracellular haloalkanes using a fluorescence-based method.
- To overcome limitations in current screening methods for enzyme discovery.
Main Methods:
- A flow cytometry-based assay utilizing catalytically deficient haloalkane dehalogenases (HaloTag-like proteins) to trap haloalkanes.
- Construction of a fluorescent sensor protein by fusing HaloTag with enhanced green fluorescent protein (eGFP).
- Validation using *Escherichia coli* cells exposed to various haloalkanes and halohydrin dehalogenase activity.
Main Results:
- Demonstrated semiquantitative detection of haloalkanes at single-cell resolution.
- Successfully separated cell populations based on haloalkane exposure.
- Showed distinct cell population separation based on halohydrin dehalogenase activity.
Conclusions:
- The developed assay concept enables the detection of intracellular haloalkanes.
- This method holds significant potential for screening and discovering novel halogenase activities.
- Advances in biocatalysis and enzyme discovery for chemical synthesis.
