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

被引：64

作者：

Kim, Min Woo ^{[1
]}

Wang, Wenjing ^{[1
]}

Sanchez, Mateo I. ^{[1
]}

Coukos, Robert ^{[1
]}

von Zastrow, Mark ^{[2
]}

Ting, Alice Y. ^{[3
,4
]}

机构：

[1] Stanford Univ, Dept Genet, Stanford, CA 94305 USA

[2] Univ Calif San Francisco, Program Cell Biol, San Francisco, CA 94143 USA

[3] Stanford Univ, Biol Genet & Chem, Stanford, CA 94305 USA

[4] Chan Zuckerberg Biohub, San Francisco, CA 94158 USA

来源：

ELIFE | 2017年 / 6卷

基金：

美国国家卫生研究院;

关键词：

YEAST 2-HYBRID SYSTEM; ETCH VIRUS PROTEASE; LIVING CELLS; COUPLED RECEPTORS; BETA(2)-ADRENERGIC RECEPTOR; CONFORMATIONAL-CHANGES; ARRESTIN RECRUITMENT; SIGNAL-TRANSDUCTION; SPLIT LUCIFERASE; IN-VIVO;

D O I：

10.7554/eLife.30233

中图分类号：

Q [生物科学];

学科分类号：

07 ; 0710 ; 09 ;

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.

引用

页数：24

共 46 条

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