Modified Transposons for Enhancing Protein Function

Multi-domain proteins are a common and essential component of the cellular proteome and contribute complex functions that single domains cannot achieve. The field of protein engineering uses sophisticated techniques to modify proteins for a given purpose, but generally focuses on single domains without consideration of the protein’s overall topology. In this work, we set out to develop tools that enable the high-throughput sampling of different domain connections. A novel method of random transposon-mediated domain insertion was devised which efficiently created large plasmid libraries of domain fusions. As a proof of concept, we made fluorescent biosensors for small molecules by inserting circularly permuted GFP into ligand-binding proteins. We also demonstrated the generality of this method by constructing a pool of functional dCas9 variants with insertions of a PDZ protein-protein interaction domain. These successful protein-engineering efforts illustrate the advanced functions possible with multi-domain constructs and how this transposon-based insertion method can facilitate the creation and study of this new class of synthetic proteins.