AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions
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DOI：
10.7554/elife.54983
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发表时间：
2020-05-11
期刊：
影响因子：
7.7
通讯作者：
Sawasaki, Tatsuya
中科院分区：
文献类型：
--
作者：
Kido, Kohki;Yamanaka, Satoshi;Sawasaki, Tatsuya
基于大肠杆菌 BirA 酶（例如 BioID (BirA*) 和 TurboID）的邻近生物素化是识别与细胞或生物体中的目标蛋白质相互作用的蛋白质的关键技术。然而，用于此目的的酶已经有了一些改进。在这里，我们展示了一种新型 BirA 酶 AirID（用于邻近依赖性生物素识别的祖先 BirA），它是使用祖先酶重建算法和宏基因组数据从头设计的。 AirID-融合蛋白（例如AirID-p53或AirID-I kappa B α）分别在体外和细胞内表明MDM2或ReIA的生物素化。 AirID-CRBN 在体外显示了 IKZF1 和 SALL4 的泊马度胺依赖性生物素化。 AirID-CRBN 基于链霉亲和素 Pull-down 测定对 CRL4(CRBN) 复合物中的内源性 CUL4 和 RBX1 进行生物素化。对稳定表达 AirID-1 kappa B α 的细胞的 LC-MS/MS 分析显示 RelA 蛋白的顶级生物素化。这些结果表明 AirID 是一种用于分析蛋白质-蛋白质相互作用的新型酶。
Proximity biotinylation based on Escherichia coli BirA enzymes such as BioID (BirA*) and TurboID is a key technology for identifying proteins that interact with a target protein in a cell or organism. However, there have been some improvements in the enzymes that are used for that purpose. Here, we demonstrate a novel BirA enzyme, AirID (ancestral BirA for proximity-dependent biotin identification), which was designed de novo using an ancestral enzyme reconstruction algorithm and metagenome data. AirID-fusion proteins such as AirID-p53 or AirID-I kappa B alpha indicated biotinylation of MDM2 or ReIA, respectively, in vitro and in cells, respectively. AirID-CRBN showed the pomalidomide-dependent biotinylation of IKZF1 and SALL4 in vitro. AirID-CRBN biotinylated the endogenous CUL4 and RBX1 in the CRL4(CRBN) complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-I kappa B alpha showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.