title: Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display.
abstract: WW domains are small β-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications.
authors: Isogenica Ltd, Little Chesterford, Essex CB10 1XL, UK.
authors: Seema
authors: S
authors: Patel
authors: Pascale
authors: P
authors: Mathonet
authors: Agnes M
authors: AM
authors: Jaulent
authors: Christopher G
authors: CG
authors: Ullman
mesh_terms: D020816:Amino Acid Motifs; D000596:Amino Acids / Q000737:chemistry*; D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D055550:Protein Stability; D059748:Proteolysis; D014364:Tryptophan / Q000737:chemistry; D014471:Umbilical Veins / Q000737:chemistry / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry* / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D020533:Angiogenesis Inhibitors; D019151:Peptide Library; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D014364:Tryptophan; D040301:Vascular Endothelial Growth Factor Receptor-2