Shrivastava A, von Wronski M A, Sato A K, Dransfield D T, Sexton D, Bogdan N, Pillai R, Nanjappan P, Song B, Marinelli E, DeOliveira D, Luneau C, Devlin M, Muruganandam A, Abujoub A, Connelly G, Wu Q L, Conley G, Chang Q, Tweedle M F, Ladner R C, Swenson R E, Nunn A D
Ernst Felder Laboratories, Bracco Research USA, Princeton, NJ 08540, USA.
Protein Eng Des Sel. 2005 Sep;18(9):417-24. doi: 10.1093/protein/gzi049. Epub 2005 Aug 8.
We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.
我们描述了一种生成受体酪氨酸激酶（RTK）高亲和力肽（HAP）结合物的全新通用方法，该方法采用多步骤流程，包括噬菌体展示筛选、鉴定适合异源二聚化（非竞争性和协同性）的肽对以及异源二聚体的化学合成。利用这一策略，我们生成了对血管内皮生长因子受体2（VEGFR-2）和c-Met的解离常数（K(D)）低于1 nM的HAP。VEGFR-2 HAP的结合显著优于用于异源二聚体合成的任何一种单个肽（6至500倍）。最重要的是，在各种竞争性结合和功能测定中，HAP作为竞争性抑制剂比天然配体（VEGF）的单体要好得多（150至800倍）。此外，我们还发现HAP的结合对血清的敏感性低于其组成肽。我们相信这种方法可应用于任何蛋白质以生成高亲和力肽（HAP）结合物。
