Automated Flow Synthesis of Combinatorial Protein Libraries

Flow synthesis with building block mixtures permits the generation of hyperdiverse polyamide libraries on a timescale of < 1 hour per library. The semi-automated flow platform on which we have previously developed this technique, howev-er, lacks the efficiency required to make protein-length polyamide chains. To facilitate the direct engraftment of diversity regions into longer polyamide chains, we adapted mixture-based library synthesis to automated flow peptide synthesis (AFPS) systems previously optimized for the production of 100-200-mer proteins. Using this approach, we demonstrate the ability to generate a 101-mer protein library with a diversity approaching quintillions of members. AFPS thus permits the incorporation of hyperdiverse polyamide libraries into synthetic protein scaffolds.