RECONSTITUTION OF SPLIT-HALOTAG VIA ORTHOGONAL TAG BINDING DOMAINS

RECONSTITUTION OF SPLIT-HALOTAG VIA ORTHOGONAL TAG BINDING DOMAINS

Researchers at UCSF have developed a method using split HaloTags to tag multiple proteins in a single assay and measure their interactions.

Due to new technologies such as CRISPR/Cas9 gene editing, it is now easier to modify DNA than at any other point in human history. One of the most common uses of this revolutionary genomic editing technology is to label proteins by genomic knock-in, specifically fluorescent proteins for identification. However, many fluorescent proteins are relatively large and can interfere with native protein localization and function. To combat this obstacle, scientists have developed short peptide tags to label cells instead, but these tags lack the fluorescent properties that enable important and commonly used techniques such as fluorescence activated cell sorting (FACS). An alternative approach is to use short split fluorescent protein (FP) tags that once expressed on a protein, can be combined with their corresponding FP fragment, and will bind to form a functional fluorescent protein. However, FP tags have intrinsic limitations that preclude their use in detecting low expression targets. In a similar vein, expanding this approach to other split protein systems is advantageous to increase the potential of these systems to be used for low-expression or high-throughput protein tagging assays. However, none of the protein splitting systems are self-complementing, limiting their usage in a strategy such as split fluorescent protein tagging.

Stage of Research

The inventors have developed a novel system for protein tagging using the existing split protein system split-Halotags. In this method, two cognate binders are bound to two unfolded split Halo fragments (nHalo and cHalo). Separately, two orthogonal peptide tags are fused to the target protein(s). These complexes are then incubated with the cognate binder/Halo enzyme complexes, and the cognate binders then bind with their matching tags. Because of the steric confinement, the two Halo fragments undergo a refolding and form a fully functional HaloTag protein. The HaloTag protein can then be used to tag proteins to allow for the assessment of a single protein’s expression (if both tags are fused to the same protein), or to assess concentrations of interacting proteins. Finally, this strategy can also be applied to different alleles in the same cell, whose concentrations can be measured in parallel.

Applications

Measuring protein-protein interactions with a fluorescence readout without compromising native protein localization
Enriching populations of knock-in cells based on fluorescence.
Sorting biallelic knock-in cells using FACS to empower use of patient-derived cellular models of genetic disease

Advantages

Small enough to label proteins fluorescently without disrupting native protein localization or interactions
This method can potentially be expanded to other split protein systems
Scalable enough to support high-throughput sorts of complicated knock-in cell populations

Stage of Development

Research- in vivo

Publications

Versatile labeling and detection of endogenous proteins using tag-assisted split enzyme complementation S. Makhija, D. Brown, R.M. Rudlaff, J. Doh, S. Bourke, Y. Wang, S. Zhou, R. Cheloor-Kovilakam, B. Huang ACS Chem. Biol. 16, 671-681 (2021) [Link] [Preprint: bioRxiv/2020.12.01.407072]

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