Engineering enhanced EGF mutants by directed evolution

表皮生长因子（EGF）在许多细胞类型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产生表皮生长因子受体（EGFR）的细胞外结构域的结合亲和力增强的EGF突变体。基本原理是，EGF突变体与不同受体亚结构域的结合亲和力增加，将引起不同的细胞反应。将产生可溶性细胞外EGFR结构域片段并用于筛选和分选通过酵母表面展示技术产生的EGF突变体文库。以高亲和力特异性结合EGFR片段的EGF突变体将以可溶形式产生，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、乳腺和卵巢肿瘤。与受体结合而不诱导活化的拮抗剂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迁移到受影响区域而参与伤口愈合。在较低剂量下引起增强功效的超激动剂EGF突变体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.