Chojnowski Alexandre, Sobota Radoslaw M, Ong Peh Fern, Xie Wei, Wong Xianrong, Dreesen Oliver, Burke Brian, Stewart Colin L
Developmental and Regenerative Biology, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.
Functional Proteomics Laboratory, Institute of Molecular & Cell Biology, A*STAR, Singapore, Singapore; Institute of Medical Biology, A*STAR, Singapore, Singapore.
iScience. 2018 Dec 21;10:40-52. doi: 10.1016/j.isci.2018.11.023. Epub 2018 Nov 15.
The modulation of protein-protein interactions (PPIs) is an essential regulatory activity defining diverse cell functions in development and disease. BioID is an unbiased proximity-dependent biotinylation method making use of a biotin-protein ligase fused to a protein of interest and has become an important tool for mapping of PPIs within cellular contexts. We devised an advanced method, 2C-BioID, in which the biotin-protein ligase is kept separate from the protein of interest, until the two are induced to associate by the addition of a dimerizing agent. As proof of principle, we compared the interactomes of lamina-associated polypeptide 2β (LAP2β) with those of lamins A and C, using 2C- and conventional BioID. 2C-BioID greatly enhanced data robustness by facilitating the in silico elimination of non-specific interactors as well as overcoming the problems associated with aberrant protein localization. 2C-BioID therefore significantly strengthens the specificity and reliability of BioID-based interactome analysis, by the more stringent exclusion of false-positives and more efficient intracellular targeting.
蛋白质-蛋白质相互作用（PPI）的调节是一种至关重要的调控活动，它决定了发育和疾病中多种细胞功能。BioID是一种无偏向性的邻近依赖性生物素化方法，利用与感兴趣的蛋白质融合的生物素-蛋白质连接酶，已成为在细胞环境中绘制PPI图谱的重要工具。我们设计了一种先进的方法，即2C-BioID，其中生物素-蛋白质连接酶与感兴趣的蛋白质保持分离，直到通过添加二聚剂诱导两者结合。作为原理验证，我们使用2C-BioID和传统BioID比较了核纤层相关多肽2β（LAP2β）与核纤层蛋白A和C的相互作用组。2C-BioID通过促进在计算机上消除非特异性相互作用分子以及克服与异常蛋白质定位相关的问题，大大增强了数据的稳健性。因此，2C-BioID通过更严格地排除假阳性和更有效地进行细胞内靶向，显著增强了基于BioID的相互作用组分析的特异性和可靠性。
