Protein-protein interactions (PPIs) underlie nearly all molecular mechanisms; hence their characterisation is crucial for understanding biological processes1. Different assays have been developed for characterising PPIs2,3. However, many fail to accurately reproduce the native environment in which studied PPIs occur or are impractical for high-throughput studies4,5.
We are developing a versatile, straightforward, and broad-spectrum method for characterising PPIs in their native cellular environment that is also suitable for high-throughput screenings in living cells. The method is based on a split Hygromycin-B-phosphotransferase (HPT) protein consisting of two inactive fragments that can reassemble into an active enzyme when fused to a pair of interacting proteins. Reassembled split HPT allows prokaryotic and eukaryotic cells encoding interacting proteins to survive under selection with the antibiotic Hygromycin-B. We have demonstrated the feasibility of our approach in proof-of-concept experiments with different protein pairs comprising weak to strong interactions to determine the method's applicability and sensitivity.
This new method overcomes many limitations of current assays, allowing the characterisation of in vivo PPIs in their native environment (prokaryotes, eukaryotes, and organelles) via a straightforward survival assay suitable for high-throughput screenings.