Division of Cell-Free Life Science, Proteo-Science Center, Matsuyama, Japan.
Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan.
Elife. 2020 May 11;9:e54983. doi: 10.7554/eLife.54983.
Proximity biotinylation based on 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-IκBα indicated biotinylation of MDM2 or RelA, 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 complex based on the streptavidin pull-down assay. LC-MS/MS analysis of cells that were stably expressing AirID-IκBα showed top-level biotinylation of RelA proteins. These results indicate that AirID is a novel enzyme for analyzing protein-protein interactions.
基于 BirA 酶（如 BioID（BirA*）和 TurboID）的邻近生物素化技术是鉴定细胞或生物体中与靶蛋白相互作用的蛋白质的关键技术。然而，用于该目的的酶已经有了一些改进。在这里，我们展示了一种新型的 BirA 酶，AirID（用于邻近依赖性生物素鉴定的祖先 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，如 AirID-p53 或 AirID-IκBα，分别在体外和细胞中指示 MDM2 或 RelA 的生物素化。AirID-CRBN 显示了体外依赖泊马度胺的 IKZF1 和 SALL4 的生物素化。AirID-CRBN 通过链霉亲和素下拉测定在 CRL4 复合物中对内源性 CUL4 和 RBX1 进行生物素化。稳定表达 AirID-IκBα 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。
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