Cellular Synthesis of Protein Catenanes

Direct cellular production of topologically complex proteins is of great interest both in supramolecular chemistry and protein engineering. We describe the first cellular synthesis of protein catenanes through the use of the p53 dimerization domain to guide the intertwining of two protein chains and SpyTag–SpyCatcher chemistry for efficient cyclization. The catenane topology was unambiguously proven by SDS‐PAGE, SEC, and partial digestion experiments and was shown to confer enhanced stability toward trypsin digestion relative to monomeric control mutants. The assembly–reaction synergy enabled by protein folding and genetically encoded protein chemistry offers a convenient yet powerful approach for creating mechanically interlocked, complex protein topologies in vivo. Let's get together: The combination of a protein dimerization domain and genetically encoded SpyTag–SpyCatcher protein chemistry enables the cellular synthesis of topologically complex protein catenanes and mechanically interlocked protein “tadpoles” through programmed posttranslational modification.