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Abstract:

Directed evolution of binding proteins

Carsten Hain, Niklas Hoffmann, Judith Kampa, Marius Schöller, Mikail Sahin, Pascal Schmidt, Marten Linder, Cassandra Königs, Bianca Frommer, Fabian Roeloffs and Sebastian Perez Knoche

iGEM Bielefeld-CeBiTec, Bielefeld University, Germany

The impact of antibodies in modern medicine is on a permanent rise but the cost and time factor as well as the immunization of animals, which die during the harvesting process, are still problematic. Therefore we want to establish an alternative using antibody-like binding proteins that are generated in vivo in E. coli. Profiting of the short generation cycle and the exponential growth of bacteria, we want to generate binding proteins in a much shorter period of time while also being more cost efficient. Furthermore, no animals have to suffer in the process by immunization. Our goal is to develop binding proteins in E. coli in a process of directed evolution that can subsequently be utilized in diagnostic techniques and target-mediated drug delivery against pathogens. Due to the ability of our system to quickly adapt to a certain target protein under evolutionary pressure it is especially useful in concern of quickly evolving and newly arising viral pathogens. The concept of our system subdivides into the following aspects: At first, we create a randomized library of binding protein sequences in bacteria to form the starting point of our project. As scaffolds for our binding proteins, we settled on both antibody mimetics (monobodies) and natural antibody fragments (nanobodies). In the next step, we use a two plasmid system in combination with a mutant of the polymerase I which leads to a higher mutation rate in the coding regions of our binding proteins, so that there is a chance for the binding proteins to adapt to the target proteins. Finally, we isolate the strains that produce the binding proteins with the highest affinity to the target protein by using an in vivo selection mechanisms: Mediated by the binding of our protein and the target, the cell in concern is granted a selective advantage directly increasing its evolutionary fitness.