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Chembiochem. 2019 Apr 15;20(8):1078-1087. doi: 10.1002/cbic.201800782. Epub 2019 Mar 12.

A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells.

Author information

1
Department of Chemistry, The Pennsylvania State University, University Park, PA, 16802, USA.
2
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA.
3
The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802, USA.

Abstract

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe-P2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophore-was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.

KEYWORDS:

fluorescence microscopy; fluorescent probes; imaging agents; protein aggregation; protein folding

PMID:
30609255
DOI:
10.1002/cbic.201800782

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