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

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.

Automated summary

Protein-protein interaction analysis is crucial for understanding protein functions. Researchers have developed a cell-free protein array technology, CF-PPiD, which uses proximity biotinylation to identify specific interacting proteins. This method allows for the biochemical identification and validation of protein interactions in cells.