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.

Evolving a Peptide: Library Platforms and Diversification Strategies

“…This method harnesses the so-called cis activity-the capacity of a DNA replication initiator protein (RepA), fused to the library peptides, to bind exclusively to the cognate template DNA [167]. CIS display has been applied for identification of binders of antibodies [167], protease-resistant peptides [168], and ligands of human vascular endothelial growth factor receptor [169].…”

Beyond the Natural Proteome

“…CIS display has been used in antibody research (Odegrip et al, 2004), peptide maturation, ligand discovery including therapeutic peptides (Mathonet et al, 2011), and engineering of small WW scaffolds (small β-sheet motifs; Patel, Mathonet, Jaulent & Ullman, 2013). CIS display technology has also been used to identify 12-mer peptides resistant to thrombin, chymotrypsin and plasma proteases (Eldridge, Cooley, Odegrip, McGregor, FitzGerald & Ullman, 2009).…”

Protein goldendoodles: Designing new proteins

“…Many groups have adopted strategies between these two extremes: focused combinatorial mutagenesis of protein-protein interfaces followed by selection/screening to identify library members with high affinity and specificity for a novel target 33,34 . For instance, Cortajarena et al 35 completely randomized seven amino acids (library size ~9×10 8 ) on the TPR2A-binding interface and selected TPR proteins that binds the C-terminal peptide of Dss1.…”