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bioRxiv · 10.1101/2023.10.29.564634

BenzoHTag, a fluorogenic self-labeling enzyme developed using molecular evolution

Lampkin, B.·Goldberg, B.·Kritzer, J.
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Abstract

Self-labeling proteins are powerful tools in chemical biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye, with the genetic specificity of a protein fusion. HaloTag7 is the most popular self-labeling protein due to its fast labeling kinetics and the simplicity of its chloroalkane ligand. Reaction rates of HaloTag7 with different chloroalkane-containing substrates is highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we report a molecular evolution system for HaloTag using yeast surface display that enables the screening of libraries up to 108 variants to improve reaction rates with any substrate of interest. We applied this method to produce a HaloTag variant, BenzoHTag, which has improved performance with a fluorogenic benzothiadiazole dye. The resulting system has improved brightness and conjugation kinetics, allowing for robust, no-wash fluorescent labeling in live cells. The new BenzoHTag-benzothiadiazole system has improved performance in live-cell assays compared to the existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. It was also found to be orthogonal to the silicon HaloTag7-rhodamine system, enabling multiplexed no-wash labeling in live cells. The BenzoHTag system, and the ability to optimize HaloTag for a broader collection of substrates using molecular evolution, will be very useful for the development of cell-based assays for chemical biology and drug development.

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Lampkin, B., Goldberg, B., Kritzer, J.. 2023-10-29. BenzoHTag, a fluorogenic self-labeling enzyme developed using molecular evolution. https://doi.org/10.1101/2023.10.29.564634

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