CellFree Sciences. Co. Ltd., 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan.
Proteo-Science Center, 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan.
Sci Rep. 2022 Jun 22;12(1):10592. doi: 10.1038/s41598-022-14872-w.
Protein-protein interaction (PPI) analysis is a key process to understand protein functions. Recently, we constructed a human protein array (20 K human protein beads array) consisting of 19,712 recombinant human proteins produced by a wheat cell-free protein production system. Here, we developed a cell-free protein array technology for proximity biotinylation-based PPI identification (CF-PPiD). The proximity biotinylation enzyme AirID-fused TP53 and -IκBα proteins each biotinylated specific interacting proteins on a 1536-well magnetic plate. In addition, AirID-fused cereblon was shown to have drug-inducible PPIs using CF-PPiD. Using the human protein beads array with AirID-IκBα, 132 proteins were biotinylated, and then selected clones showed these biological interactions in cells. Although ZBTB9 was not immunoprecipitated, it was highly biotinylated by AirID-IκBα, suggesting that this system detected weak interactions. These results indicated that CF-PPiD is useful for the biochemical identification of directly interacting proteins.
蛋白质-蛋白质相互作用（PPI）分析是理解蛋白质功能的关键过程。最近，我们构建了一种人类蛋白质微阵列（20K 人类蛋白珠微阵列），其中包含 19712 种由小麦无细胞蛋白生产系统产生的重组人类蛋白。在这里，我们开发了一种基于邻近生物素化的无细胞蛋白微阵列技术用于 PPI 鉴定（CF-PPiD）。邻近生物素化酶 AirID 融合的 TP53 和 -IκBα 蛋白在 1536 孔磁板上分别对特定的相互作用蛋白进行生物素化。此外，使用 CF-PPiD 显示 AirID 融合的 cereblon 具有药物诱导的 PPIs。使用带有 AirID-IκBα 的人类蛋白珠微阵列，有 132 种蛋白被生物素化，然后选择的克隆在细胞中显示出这些生物学相互作用。尽管 ZBTB9 没有被免疫沉淀，但它被 AirID-IκBα 高度生物素化，表明该系统检测到了弱相互作用。这些结果表明 CF-PPiD 可用于直接相互作用蛋白的生化鉴定。
