Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells.-猫眼课题宝

DOI：

10.1038/s42003-022-03604-5

发表时间：

2022-07-04

期刊：

Communications biology

影响因子：

5.9

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9253107/

近距离依赖性生物素化（PDB）结合质谱分析技术是研究活细胞中蛋白质相互作用的关键技术。BioID是一种广泛使用的方法，它使用了E. coliBirA生物素蛋白连接酶，一种具有缓慢标记动力学的相当大的酶。为了提高PDB的通用性和速度，已经通过不同的方法开发了各种酶。在此我们介绍了一种小型工程酶：ultraID。我们展示了其在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，我们还利用ultraID提供了一个与膜相关的coatomer相互作用体，即COPI囊泡的外壳蛋白复合物。到目前为止，ultraID是可用于PDB的最小和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体的可能性。本发明提供了一种用于邻近依赖性生物素化的小尺寸工程酶，ultraID，其在哺乳动物细胞培养物，E. coli和革兰氏阳性菌S.啤酒。

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. A small-size engineered enzyme, ultraID, is presented for proximity-dependent biotinylation, that shows efficient labeling in mammalian cell culture, E. coli and S. cerevisiae.