The ability to generate high-affinity and high-stability peptide ligands is critical in the production of robust biosensors, stable peptide therapeutics, and highly specific imaging reagents. Kalata B1 (KB1) is a cyclic cystine knot peptide that resists enzymatic, thermal, and chemical denaturation, and acyclic permutants have been shown to retain these properties. KB1 was investigated as a peptide scaffold in an effort to generate low molecular weight and enzymatically stable affinity ligands. Using bacterial cell surface display, a large library of variants was constructed by randomizing seven residues within the KB1 scaffold. The library was then sorted to identify peptides that bind specifically to human thrombin. From the selected clones, several distinct consensus groups were identified and the peptides were ranked by their dissociation rate. Thrombin-specific peptide variants were purified and characterized using RP-HPLC and mass spectrometry. The peptides exhibited equilibrium dissociation constants in the high nanomolar range as measured using surface plasmon resonance. To determine if the thrombin-binding KB1 peptides retain the stability of the original scaffold, their resistance to trypsin and chymotrypsin proteolysis and to heat denaturation was measured. Our results indicate that the KB1 scaffold may be useful for creating high-stability affinity ligands.

Extended Abstract Status: Not Uploaded