Time-gated detection of protein-protein interactions with transcriptional readout

eLife

Alice Y Ting¹  Robert Coukos¹  Mateo I Sanchez¹  Wenjing Wang¹  Min Woo Kim¹  Mark von Zastrow² 

[1] Department of Genetics, Stanford University, Stanford, United States;Program in Cell Biology, University of California, San Francisco, San Francisco, United States;

关键词: proximity-dependent labeling;    Tango;    TEV protease;    genetic selection;    GPCR;    LOV domain;   

DOI  :  10.7554/eLife.30233

来源: DOAJ

【 摘 要 】

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.

【 授权许可】

Unknown