Genome-wide physical protein–protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.

全基因组物理蛋白质-蛋白质相互作用(PPI) 作图仍然是当前技术面临的主要挑战。在这里，我们报道了一种高效的 BiFC-seq 方法，酵母增强的绿色荧光蛋白基双分子荧光互补(yEGFP-BiFC) 与下一代 DNA 测序相结合，用于相互作用组映射。我们首先应用 yEGFP-BiFC 方法系统地研究埃博拉病毒的病毒内网络。三分之二 (9/14) 的已知 EBOV 相互作用被重新捕获，并发现了五种新的相互作用。接下来，我们使用 BiFC-seq 方法绘制了肿瘤蛋白 p53 的相互作用组图。我们确定了 97 个 p53 相互作用因子，其中超过四分之三是新的。此外，在更复杂的背景下，我们通过将两个 BiFC 文库汇集在一起​​来筛选潜在的相互作用者，并揭示了 205 种蛋白质之间的 229 种相互作用的网络。这些结果表明，BiFC-seq 是一种高度灵敏、快速且经济的全基因组相互作用组作图方法。