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Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system
Nucleic Acids Research ( IF 16.6 ) Pub Date : 2017-11-20 , DOI: 10.1093/nar/gkx1173
Fang Yang 1, 2 , Yingying Lei 3, 4 , Meiling Zhou 3, 4 , Qili Yao 3, 4 , Yichao Han 5 , Xiang Wu 3, 4 , Wanshun Zhong 1, 2 , Chenghang Zhu 3, 4 , Weize Xu 3, 4 , Ran Tao 3, 4 , Xi Chen 3, 4 , Da Lin 3, 4 , Khaista Rahman 3, 4 , Rohit Tyagi 3, 4 , Zeshan Habib 3, 4 , Shaobo Xiao 3, 4 , Dang Wang 3, 4 , Yang Yu 6 , Huanchun Chen 3, 4 , Zhenfang Fu 3, 4, 7 , Gang Cao 3, 4, 5, 8, 9
Affiliation  

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based ‘library vs library’ Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome–lysosome fusion. This concept can also be applied to other systems to screen protein–RNA and protein–DNA interactions and delineate signaling landscape in cells.

中文翻译:


基于重组的文库与文库高通量酵母双杂交(RLL-Y2H)筛选系统的开发和应用



蛋白质-蛋白质相互作用(PPI)网络维持所有生物体的正常功能。迫切需要简单的高通量技术来描绘 PPI 网络的景观。虽然最近最先进的酵母双杂交(Y2H)系统提高了筛选效率,但仍然需要单个菌落分离、文库制备阵列、基因条形码或大规模测序。在这里,我们开发了一种基于重组的“文库与文库”Y2H系统(RLL-Y2H),通过该系统可以在单个库中完成多文库筛选,而无需任何单独处理。该系统基于 phiC31 整合酶介导的诱饵和猎物质粒之间的整合。整合的片段被Mmel消化并进行深度测序以解码相互作用矩阵。我们应用该系统破译了结核分枝杆菌与宿主细胞之间的跨界相互作用组,并进一步鉴定了干扰吞噬体-溶酶体融合的 Rv2427c。这个概念也可以应用于其他系统,以筛选蛋白质-RNA 和蛋白质-DNA 相互作用并描绘细胞中的信号传导景观。
更新日期:2017-11-20
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