Peptide agonists and antagonists represent a rapidly growing segment among biological therapeutics. Their small size can beneficially improve tissue penetration, and enable modular integration with other therapeutic molecules, imaging agents, and nanotechnologies. Previously, phage display technology was applied to identify and affinity mature peptides that bind vascular endothelial growth factor (VEGF), through multiple cycles of diversification and selection [1]. To investigate the utility of bacterial display in affinity maturation, we devised a simple strategy to favor high-affinity VEGF-binding ligands by their ability to compete with a decoy protein of known binding affinity. A consensus motif of W-E/D-W-E/D was initially discovered to confer binding to VEGF and was further improved through two rounds of affinity maturation. In addition, a biterminal display scaffold was used to normalize for peptide expression level; VEGF-binding peptides were displayed on the N-terminus of eCPX [2], and an expression normalization tag was fused to the C-terminus. This allowed the apparent affinity of each peptide to be decoupled from the number of peptides displayed on each bacterial clone and enhanced the differences in fluorescence profiles between high- and low-affinity ligands. A large family of highly related VEGF-binding peptides was identified, and some clones exhibited high similarity with the best VEGF binder identified previously using phage display. Peptide affinities were then measured using surface plasmon resonance to establish a relationship between cell surface ranked affinities and solution affinities.

1. Fairbrother, W.J., et al., Novel peptides selected to bind vascular endothelial growth factor target the receptor-binding site. Biochemistry 37, 17754-17764 (1998).

2. Rice, J.J. & Daugherty, P.S. Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Engineering, Design and Selection 21, 435-442 (2008).

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