Array for identification of protein-protein interactions

Protein arrays have become a valuable tool in high-throughput biology. We have developed a rapid, sensitive and widely applicable array-based technology for the screening of human protein-protein interactions using bacterial lysates. Crude protein extracts were prepared from 13,824 E. coli expression clones, high-density spotted onto filter membranes and screened for their ability to interact with unpurified recombinant human proteins. Using a filter-based enzyme-linked immunosorbent assay, novel interaction pairs such as CHIP/caytaxin, amphiphysin II/DLP4 or VCP/AMFR were identified, and subsequently confirmed by pull-down, two-hybrid and functional assays. Moreover, binding motifs in the amphiphysin II interacting proteins DLP4, XRCC4 and FBP were mapped using peptide arrays. By eliminating the need for protein purification, arrays of crude protein extracts can now be utilized successfully for the identification of protein-protein interactions and might also be applicable for systematic protein-drug and protein-DNA interaction screens.