Synthesis of epitope-targeting nanobody based on native protein-protein interactions for FtsZ filamentation suppressor.

Phage display and biopanning are powerful tools for generating binding molecules for a specific target. However, the selection process based only on binding affinity provides no assurance for the antibody's affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein-protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the nanobody, and a nonrelated CDR loop (in the grafted nanobody) was randomized to create a phage display library. In this construction of nanobodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence added functionality to the nanobody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein-protein interactions. To apply for biological tool with growth screening, model nanobodies with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) from Staphylococcus aureus were constructed and completely inhibited the polymerization of FtsZ as a function. Consequently, the expression of these nanobodies drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein-protein interactions for steady epitope-specific evolutionary engineering.