期刊
CHEMBIOCHEM
卷 20, 期 8, 页码 1078-1087出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201800782
关键词
fluorescence microscopy; fluorescent probes; imaging agents; protein aggregation; protein folding
资金
- Burroughs Wellcome Fund Career Award at the Scientific Interface
- Paul Berg Early Career Professorship
- Lloyd and Dottie Huck Early Career Award
- Sloan Research Fellowship
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 probeP2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophorewas 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据