Engineering enhanced EGF mutants by directed evolution

表皮生长因子（Epidermal growth factor， EGF）在多种细胞的增殖和分化中起着重要作用。本提案的目标是使用定向进化来产生与表皮生长因子受体（EGFR）细胞外结构域结合亲和力增强的EGF突变体。其基本原理是，与不同受体亚域结合亲和力增加的EGF突变体将引起不同的细胞反应。将生成可溶性细胞外EGFR结构域片段，并用于筛选和分类酵母表面显示技术产生的EGF突变文库。高亲和力特异性结合EGFR片段的EGF突变体将以可溶性形式产生，并测试其调节生物功能的能力。EGFR在许多细胞上过度表达和/或组成性激活。肿瘤包括脑瘤、乳腺瘤和卵巢瘤。与受体结合而不诱导激活的拮抗EGF配体的产生可能具有有效的癌症治疗应用。此外，EGF通过促进成纤维细胞增殖和向患处迁移而参与伤口愈合。超激动剂EGF突变体在较低剂量下可增强功效，可用于组织工程应用。

Epidermal growth factor (EGF) plays an important role in the proliferation and differentiation of many cell types. The goal of this proposal is to use directed evolution to generate EGF mutants with enhanced binding affinities to the extracellular domains of the epidermal growth factor receptor (EGFR). The rationale is that EGF mutants with increased binding affinity to different receptor subdomains will elicit distinct cellular responses. Soluble extracellular EGFR domain fragments will be generated and used to screen and sort EGF mutant libraries produced by yeast surface display technologies. EGF mutants that specifically bind to the EGFR fragments with high affinity will be produced in soluble form, and tested for their ability to modulate biological functions. The EGFR is over-expressed and/or constitutively activated on many. tumors, including brain, mammary and ovarian. The generation of antagonist EGF ligands that bind to the receptor without inducing activation could have potent cancer therapeutic applications. Additionally, EGF has been implicated in wound healing through promotion of fibroblast proliferation and migration to the affected area. Superagonist EGF mutants that elicit enhanced efficacy at lower doses could be useful in tissue engineering applications.