Dual surface selection methodology for the identification of thrombin binding epitopes from hotspot biased phage-display libraries

Protein libraries biased towards amino-acid residues found at so-called 'hotspots' were incorporated into the beta-sheet region of the thermostable variant (HTB1) of the B1 domain of the immunoglobulin (IgG) binding protein G and expressed as gene 3 fusions on M13 bacteriophage. The HTB1 library (2.2×10⁹) variants with a minimal 12 amino acid basis set were selected for binding IgG, to ensure structural conservation, and subsequently to thrombin to evolve a thrombin-binding function. We believe that this dual surface selection strategy will have great utility in evolving new bi-functional proteins without compromising structure. Furthermore the discrete beta-sheet epitopes identified by our methodology will lend itself to small-molecule mimicry of beta-sheets.