LAUSR

Semisynthetic cyclic peptides bearing both non-proteinogenic and genetically encoded amino acids are excellent ligands for peptide-based drug discovery. While semisynthesis expands the chemical space, genetic encoding allows access to a large library via randomization at the nucleic acid level. Selection of novel binders of such macrocyclic ligands requires linking their genotype to phenotype. In this chapter, we report a bacterial cell-surface display system to present cyclic peptides composed of synthetic and genetically encoded fragments. The synthetic fragment along with the split intein partner and an aminooxy moiety is ligated and cyclized with the recombinant backbone containing an unnatural amino acid by protein trans-splicing and intramolecular oxime ligation, respectively. A pH-shift protocol was applied to accelerate on surface cyclization. This method will enable generation of semisynthetic cyclic peptide libraries and their selection by fluorescence-activated cell sorting.