Isogenica Ltd, Little Chesterford, Essex CB10 1XL, UK.
Protein Eng Des Sel. 2013 Apr;26(4):307-15. doi: 10.1093/protein/gzt003. Epub 2013 Feb 1.
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.
WW 结构域是小的β-折叠基序，通过识别富含脯氨酸或磷酸化的线性肽序列，参与细胞内信号转导。在这里，我们描述了对该结构域的修饰，为工程化其凹面暴露的侧链提供了一个框架。这个非天然支架包含一个额外的色氨酸，具有更短的环 1，并且支持对 25%的天然蛋白质进行修饰，以形成一种新型亲和试剂。我们通过使用无细胞分子展示平台 CIS 展示，从修饰文库中选择与人血管内皮生长因子受体同种型 2（VEGFR-2）的细胞外区域具有高亲和力结合物，证明了该结构的实用性。该分离物对 VEGFR-2 的亲和力为纳摩尔级，并以纳摩尔级的活性抑制人 VEGF 与其受体的结合。该结构适合环化以提高其蛋白酶稳定性，并且与较大的蛋白质支架相比具有优势，因为它可以通过化学方法以高产率合成，为治疗和非治疗应用提供了潜力。
