Department of Genetics, Stanford University, Stanford, United States.
Program in Cell Biology, University of California, San Francisco, San Francisco, United States.
Elife. 2017 Nov 30;6:e30233. doi: 10.7554/eLife.30233.
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 a PPI to deliver a protease proximal to its cleavage peptide 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.
转录分析，如酵母双杂交和 TANGO，可将瞬时蛋白-蛋白相互作用（PPIs）转化为转基因的稳定表达，是发现 PPI、筛选和分析细胞群体的有力工具。然而，这种分析通常具有较高的背景，并且会丢失关于 PPI 动态的信息。我们开发了 SPARK（具有快速动力学的特定蛋白关联工具，可提供转录读数），其中膜锚定转录因子（TF）的蛋白水解释放需要 PPI 将蛋白酶递送到其切割肽附近，然后用蓝光去封闭切割位点。SPARK 用于检测哺乳动物细胞中的 12 种不同的 PPIs，具有 5 分钟的时间分辨率和高达 37 的信号比。通过移动光窗，我们可以重建 PPI 时程。与 FACS 结合使用时，SPARK 可以使 PPI 阳性细胞相对于 PPI 阴性细胞富集 51 倍。由于其高特异性和灵敏度，SPARK 有可能推进 PPI 分析和发现。
