Cell-free cotranslation and selection using in vitro virus for high-throughput analysis of protein–protein interactions and complexes

Abstract

We have developed a simple and totally in vitro selection procedure based on cell-free cotranslation using a highly stable and efficient in vitro virus (IVV). Cell-free cotranslation of tagged bait and prey proteins is advantageous for the formation of protein complexes and allows high-throughput analysis of protein–protein interactions (PPI) as a result of providing in vitro instead of in vivo preparation of bait proteins. The use of plural selection rounds and a two-step purification of the IVV selection, followed by in vitro post-selection, is advantageous for decreasing false positives. In a single experiment using bait Fos, more than 10 interactors, including not only direct, but also indirect interactions, were enriched. Further, previously unidentified proteins containing novel leucine zipper (L-ZIP) motifs with minimal binding sites identified by sequence alignment as functional elements were detected as a result of using a randomly primed cDNA library. Thus, we consider that this simple IVV selection system based on cell-free cotranslation could be applicable to high-throughput and comprehensive analysis of PPI and complexes in large-scale settings involving parallel bait proteins.