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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Improving split-HaloTag through computational protein engineering.

Jonas Wilhelm1, Lennart Nickel1, Yin-Hsi Lin1

  • 1Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg, Germany.

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Researchers improved the split-HaloTag system for recording cellular events. Engineered protein fragments enhance labeling speed and sensitivity, enabling faster and more accurate molecular recording in cells.

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PROSSRosettaSplit‐HaloTagcircular permutationfluorescencemolecular recorderprotein designprotein engineeringprotein labeling

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cellular Imaging

Background:

  • Split-HaloTag technology enables recording transient molecular interactions as permanent cellular marks.
  • Current limitations include slow labeling rates, restricting the application of split-HaloTag recorders.

Purpose of the Study:

  • To engineer an improved version of cpHalo∆, the larger fragment of the split-HaloTag system, to enhance labeling kinetics.
  • To increase the thermostability and activity of the cpHalo∆ fragment for broader applications.

Main Methods:

  • Computational techniques were employed to identify stabilizing point mutations in cpHalo∆.
  • A structured linker was designed to connect the N and C termini of the circular permutated protein.
  • The engineered cpHalo∆ variant was characterized for thermostability and labeling activity.

Main Results:

  • The engineered cpHalo∆ demonstrated significantly improved thermostability and activity compared to the wild-type.
  • The modified system requires lower substrate concentrations and reduced labeling times.
  • Enhanced dynamic range and sensitivity were observed in split-HaloTag-based assays.

Conclusions:

  • The engineered cpHalo∆ fragment represents a significant advancement in split-HaloTag technology.
  • This improvement facilitates faster and more sensitive recording of transient molecular events in cells.
  • The enhanced system expands the utility of split-HaloTag recorders in various biological research areas.