We developed a novel and easy to use system for the generation of binding proteins in E. coli via in vivo directed evolution. Resulting proteins called Evobodies have the potential to bind specific to target proteins enabling varies medical and analytical applications. Big advantages of our low-cost system are the short hands on time and the short generation time.

As the starting point of our system, we designed and synthesized a library of a new entity of BioBricks encoding binding proteins in E. coli based on Nanobodies as well as Monobodies. The diversity of the library in E. coli is continuously increased by co-expressing a special DNA-Polymerase conferring plasmid restricted error-prone replication of the binding protein expressing plasmids. Finally, binding proteins with high affinity to the target protein are selected using a bacterial two-hybrid system providing growth advantage to antibiotics in relation to protein-protein interaction strength. Ultimately, desired clones are enriched during fermentation in batch or in continuous culture.

Binding capacities of our libraries, efficiency of our selection system and potential of our mutagenesis system were demonstrated. Moreover, library diversity and mutation system characteristics were analyzed in detail by high-throughput sequencing.