title: A 45-Amino-Acid Scaffold Mined from the PDB for High-Affinity Ligand Engineering.
abstract: 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 10(8) 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.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Max A
authors.initials: MA
authors.lastname: Kruziki
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Sumit
authors.initials: S
authors.lastname: Bhatnagar
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Woldring
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Vandon T
authors.initials: VT
authors.lastname: Duong
authors.affiliation: Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: hackel@umn.edu.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hackel
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D030562:Databases, Protein; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D008024:Ligands; C512478:EGFR protein, human; D066246:ErbB Receptors