Resistance-based directed evolution of nanobodies for higher affinity in prokaryotes

A prokaryotic resistance-based directed evolution system leveraging protein-fragment complementation assay (PCA) was devised, and its proficiency in detecting protein-protein interactions and discriminating varying degrees of binding affinity was demonstrated by two well-characterized protein pairs. Furthermore, we constructed a random mutant library based on the GBP^R36K/E45K mutant, characterized by almost no affinity towards EGFP. This library was subjected to PCA-based prokaryotic directed evolution, resulting in the isolation of back-mutated variants. In summary, we have established an expedited, cost-effective, and structural information-independent PCA-based prokaryotic directed evolution platform for nanobody affinity maturation, featuring tunable screening stringency via modulation of antibiotic concentrations.