Phage selection of bicyclic peptides based on two disulfide bridges

Bicyclic peptides can bind with high affinity and selectivity to protein targets, making this format attractive for biotechnological and medicinal applications. The good binding properties are based to a large extent on the limited conformational flexibility of the two connected peptide rings. Bicyclic peptides with desired binding specificity can be isolated from phage display libraries that are generated by chemically cyclizing linear peptide on phage with alkylating reagents. Recently, we presented a strategy for the phage selection of bicyclic peptides based on two disulfide bridges. This approach allows the generation and screening of topologically highly diverse bicyclic peptide structures. Herein, we describe step-by-step protocols to clone and produce disulfide-cyclized bicyclic peptide libraries as well as to screen the libraries and to synthesize and characterize isolated bicyclic peptides.