应用定向进化获取受体高亲和性hEGF突变株E40V及其特性的评价

Identification of human EGFR high-affinity mutant E40V via directed evolution in vitro

人表皮生长因子(human epidermal growth factor,hEGF)是一种由53个氨基酸构成的单体多肽。该蛋白作为有效的促有丝分裂因子,通过和细胞表面受体EGFR特异性结合来影响细胞的生长,增殖和分化。hEGF基因的失调与某些癌症的生长和发育有关,其蛋白本身对于表皮损伤和溃疡具有良好的修复功能。但是由于其半衰期较短且会造成受体表达下调,因此有必要对其进行改良。通过StEP法对人源和猪源EGF因子进行定向化改造,通过构建噬菌体展示文库和ELISA筛选方式获得一株对于受体具有较高亲和力的突变株E40V,并使用MTT、圆二色谱和细胞划痕对该突变株的促细胞增殖,迁移能力以及温度,pH耐受性进行了探究。结果显示,E40V相较于野生型EGF,其受体亲和性显著提高,对于正常细胞的促增殖能力也有所强化,而对于温度,pH耐受情况则与野生型相差不大。本研究不仅为短肽的定向进化提供了一个范例,也为EGF的实际应用打下基础。

Human epidermal growth factor(hEGF)is a monomeric polypeptide consisting of 53 amino acids.The protein acts as an active mitogenic factor and affects cell growth,proliferation and differentiation by specifically binding to its cell surface receptor EGFR.The dysregulation of the hEGF gene is associated with the growth and development of certain cancers.Although hEGF protein shows a quick repair function for epidermal tissue damage and ulceration disease,some ambiguities such as short half-life and EGFR down-regulation still remain.In this study,the human EGF and porcine EGF factor were modified via directed evlotion in vitro by the staggered extension process(StEP),in which a mutant E40V with high affinity against EGFR receptor was obtained using phage display library construction and ELISA screening.Then we used MTT,circular dichroism(CD)and wound healing assay to analyze the A431 and NIH3T3 cell proliferation,migration ability,temperature and pH tolerance of the mutant protein respectively.We found that the affinity of E40V for EGFR was significantly higher than that of wild-type EGF and the mutant could also promote the proliferation of normal cells NIH3T3,while the temperature tolerance and pH tolerance were similar to those of wild-type.Our DNA shuffling EGF with targeting properties and long half-life may provide a new application foreground for regulations of the normal cell growth and cancer targeting therapy.