Summary Small protein ligands can provide superior physiological distribution compared with antibodies, and improved stability, production, and specific conjugation. Systematic evaluation of the PDB identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α helix opposite a β sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 108 mutants and directed evolution toward four targets yielded target-specific binders with affinities as strong as 200 ± 100 pM, Tms from 65°C ± 3°C to 80°C ± 1°C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ± 8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold.
Bibliographical noteFunding Information:
We are grateful to Dr. Sivaramesh Wigneshweraraj for the JE1 E. coli strain and Aaron Becker at the University of Minnesota Genomics Center for assistance with Illumina sequencing. This work was partially funded by the Department of Defense (Grant W81XWH-13-1-0471 to B.J.H.), the NIH (Grant EB019518 to B.J.H.), and the University of Minnesota.
© 2015 Elsevier Ltd.