TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation.

The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TAT-dependent translocation of the resistance marker TEM β-lactamase (βL). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and βL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the βL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split βL fragments and TorA, respectively.