CF-PPiD technology based on cell-free protein array and proximity biotinylation enzyme for in vitro direct interactome analysis

被引：3

作者：

Sugiyama, Shusei ^{[1
]}

Yamada, Kohdai ^{[2
]}

Denda, Miwako ^{[1
]}

Yamanaka, Satoshi ^{[2
]}

Ozawa, Satoshi ^{[1
]}

Morishita, Ryo ^{[1
]}

Sawasaki, Tatsuya ^{[2
]}

机构：

[1] CellFree Sci Co Ltd, 3 Bunkyo Cho, Matsuyama, Ehime 7908577, Japan

[2] Proteosci Ctr, 3 Bunkyo Cho, Matsuyama, Ehime 7908577, Japan

来源：

SCIENTIFIC REPORTS | 2022年 / 12卷 / 01期

基金：

日本学术振兴会;

关键词：

NF-KAPPA-B; DEGRADATION; TARGET; PHOSPHORYLATION; IDENTIFICATION; THALIDOMIDE; CEREBLON; PROMOTES; RECEPTOR; BINDING;

D O I：

10.1038/s41598-022-14872-w

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 -I kappa B alpha 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-I kappa B alpha, 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-I kappa B alpha, suggesting that this system detected weak interactions. These results indicated that CF-PPiD is useful for the biochemical identification of directly interacting proteins.

引用

页数：14