| Autor: |
Lampkin BJ; Department of Chemistry, Tufts University, Medford MA 02155., Goldberg BJ; Department of Chemistry, Tufts University, Medford MA 02155., Kritzer JA; Department of Chemistry, Tufts University, Medford MA 02155. |
| Jazyk: |
angličtina |
| Zdroj: |
BioRxiv : the preprint server for biology [bioRxiv] 2024 Apr 02. Date of Electronic Publication: 2024 Apr 02. |
| DOI: |
10.1101/2023.10.29.564634 |
| Abstrakt: |
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 10 8 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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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