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

Abstract

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.

GATS tag system is compatible with biotin labelling methods for protein analysis

“…Polypeptide tag technology is used in many PPI analyses, such as co-immunoprecipitation 8 , AlphaScreen assay 9 – 11 , and protein array 12 – 14 . Tag systems are also used in many life science experiments, such as cell biology and transgenic organisms.…”

GATS Tag System for Protein Analysis with Biotin Labelling Methods

“…Polypeptide tag technology is used in many PPI analyses, such as coimmunoprecipitation 8 , AlphaScreen assay 9,10,11 , and protein array 12,13,14 . Tag systems are also used in many life science experiments, such as cell biology and transgenic organisms.…”