Communications Biology ( IF 5.2 ) Pub Date : 2022-07-04 , DOI: 10.1038/s42003-022-03604-5
Lea Kubitz 1 , Sebastian Bitsch 2 , Xiyan Zhao 1 , Kerstin Schmitt 3 , Lukas Deweid 2, 4 , Amélie Roehrig 1, 5 , Elisa Cappio Barazzone 1, 6 , Oliver Valerius 3 , Harald Kolmar 2 , Julien Béthune 7
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Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.
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UltraID 的工程,一种紧凑且高度活跃的酶,用于活细胞中的邻近依赖性生物素化
邻近依赖性生物素化 (PDB) 结合质谱分析已成为研究活细胞中蛋白质-蛋白质相互作用的关键技术。一种广泛使用的方法 BioID 使用大肠杆菌的无效变体BirA 生物素蛋白连接酶,一种非常庞大的酶,具有缓慢的标记动力学。为了提高 PDB 的多功能性和速度,已经通过不同的方法开发了各种酶。在这里,我们展示了一种小型工程酶:ultraID。我们展示了它在 10 分钟标记脉冲和生理水平表达后探测 Argonaute-2 相互作用组的实际用途。此外,使用ultraID,我们提供了一种与膜相关的涂层相互作用组,即COPI囊泡的外壳蛋白复合物。迄今为止,ultraID 是可用于 PDB 的最小且最有效的生物素连接酶,并提供了以高时间分辨率研究相互作用组的可能性。