Co-translation to detect ultraweak protein-protein interactions

Most cellular processes take place by protein complexes. Many of these activities occur in transient complexes, therefore ultraweak interactions are necessary to regulate these dynamic interactions. In bacteria, protein complexes are co-expressed in operons from a single mRNA in a co-transcriptional and co-translational manner. This facilitates their interactions and accelerates the biochemical process. We harnessed this phenomenon to study eukaryotic ultraweak protein-protein interactions (PPIs) which are difficult to study. We developed a novel genetic selection system in bacteria, based on split-chloramphenicol-acetyl transferase (Split-CAT). In this system, two potential proteins of interest are each fused to the split-CAT fragments and are co-expressed from a single RNA in E. coli. Upon binding, the two CAT fragments are functionally assembled, leading to bacterial survival on selective media containing chloramphenicol. We demonstrated that expression of ultraweak PPIs from two different vectors did not yield growth, however, co-translation of the same PPIs from a single vector resulted in significant selective growth in media supplemented with chloramphenicol. We employed the system to identify novel ubiquitin binding proteins and characterized their interfaces using a rich library of ubiquitin mutants.