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BenzoHTag, a fluorogenic self-labeling protein developed using molecular evolution.

Bryan J Lampkin¹, Benjamin J Goldberg¹, Joshua A Kritzer¹

¹Department of Chemistry, Tufts University, Medford MA 02155.

Biorxiv : the Preprint Server for Biology

|April 15, 2024

Summary

Researchers developed a new molecular evolution system to improve self-labeling proteins like HaloTag. This led to BenzoHTag, enabling faster, brighter live-cell labeling with benzothiadiazole dyes for chemical biology applications.

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Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

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Published on: December 21, 2017

Area of Science:

Chemical Biology
Molecular Biology
Biotechnology

Background:

Self-labeling proteins, such as HaloTag7, facilitate precise cellular localization of synthetic molecules via genetic protein fusions.
HaloTag7's utility is limited by slow labeling kinetics with many substrates, hindering live-cell assay applications.
Fast labeling rates are crucial for effective live-cell assays in chemical biology.

Approach:

Developed a molecular evolution system using yeast surface display to screen large libraries (up to 10^8 variants) of HaloTag.
Engineered a novel HaloTag variant, BenzoHTag, optimized for improved reaction rates with specific substrates.
Focused on enhancing performance with fluorogenic benzothiadiazole dyes.

Key Points:

The BenzoHTag-benzothiadiazole system demonstrates enhanced brightness and conjugation kinetics for robust, no-wash fluorescent labeling in live cells.
Achieved rapid intracellular enzyme saturation (<100 seconds) and effective labeling at low dye concentrations (7 nM).
BenzoHTag proved orthogonal to the HaloTag7-silicon rhodamine system, enabling multiplexed labeling.

Conclusions:

The molecular evolution approach significantly enhances HaloTag performance with diverse substrates.
BenzoHTag offers superior performance in live-cell assays compared to existing HaloTag systems.
This technology advances cell-based assay development for chemical biology and drug discovery.