Abstract
A general method was developed for the discovery of protease-activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease-2 substrate linker to a vascular endothelial growth factor-binding peptide and sorted using a two-stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease-mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100-fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.