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

Authors
  • Kim, Min Woo1
  • Wang, Wenjing1
  • Sanchez, Mateo I1
  • Coukos, Robert1
  • von Zastrow, Mark2
  • Ting, Alice Y1, 3, 4, 5
  • 1 Department of Genetics, Stanford University, Stanford, United States. , (United States)
  • 2 Program in Cell Biology, University of California, San Francisco, San Francisco, United States. , (United States)
  • 3 Department of Biology, Stanford University, Stanford, United States. , (United States)
  • 4 Department of Chemistry, Stanford University, Stanford, United States. , (United States)
  • 5 Chan Zuckerberg Biohub, San Francisco, United States. , (United States)
Type
Published Article
Journal
eLife
Publisher
"eLife Sciences Organisation, Ltd."
Publication Date
Nov 30, 2017
Volume
6
Identifiers
DOI: 10.7554/eLife.30233
PMID: 29189201
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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.

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