噬菌体展示非天然多元环肽库的设计合成与蛋白识别分子筛选

There are a number of mini-protein and multiple cyclic peptide scaffolds in nature. These scaffolds can be used in combination with screening technologies such as phage display, and this has become a very promising strategy for developing protein affinity reagents. Natural cystine knots are interesting mini-protein or multi-cyclic peptide scaffolds, and they have advantages of high structural anti-proteolytic stability. However, this kind of mini-protein also has some disadvantages, including the precise disulfide pairing and disulfide isomerization problem, and the low structural diversity. And traditional phage display technology usually suffers from difficulty of expressing unnatural peptides and low efficiency of post-translational modifications. Thus, in this project, we will develop unnatural multi-cyclic peptide libraries with unique and stable structures, size- and structure-tunable unnatural scaffolds, and a high degree of structural complementary to target proteins and tolerance to sequence manipulations. Moreover, we will develop bioactive peptides with a high bind affinity and specificity and a high anti-proteolytic stability for important targeting proteins that are closely relevant to human diseases or key physiological processes. We will further perform studies on the modulation of protein function and imaging analysis of target proteins by taking advantage of the developed multi-cyclic peptide affinity reagents.

自然界拥有许多迷你蛋白和多元环肽分子骨架；其与噬菌体展示等筛选技术的有机结合，已成为一种发展新型蛋白识别分子的有效手段。天然“胱氨酸结”具有构型稳定、抗酶水解能力强等优点，是一类极具前景的迷你蛋白或多元环肽分子骨架。然而，天然“胱氨酸结”多元环肽骨架存在二硫键配对精准度差、易于重排异构、缺少结构多样性等缺陷；而传统的噬菌体展示技术也受难以表达非天然序列、翻译后修饰效率低等问题限制。因此，本项目拟通过噬菌体表面天然化学连接反应和多肽分子内二硫键精准配对技术的巧妙结合，发展构型独特且稳定、非天然骨架尺寸和构型可调、与靶标蛋白识别作用区构型高度互补以及耐受序列任意调控的非天然多元环肽库，并针对与疾病或重要生理过程密切相关的重要蛋白质分子，筛选并设计具有高亲合力、高特异性和高抗酶解稳定性的非天然多元环肽识别分子。进一步以多肽识别分子为分子探针，开展靶标蛋白功能调控和成像分析等研究。

富含二硫键多肽是一类具有稳定三维结构的迷你蛋白质分子，具有成为分子探针工具和药物分子的巨大潜能。然而，目前几乎所有的富含二硫键多肽都来自于自然界，从头设计和筛选具有活性的富含二硫键多肽仍面临诸多挑战。本项目主要发展了系列不同的多肽氧化折叠调控方法，获得了各类耐受序列调控的富含二硫键多元环肽多肽骨架模板。在此基础上，本项目发展了多种新型富含二硫键多肽库，筛选获得了具有靶标蛋白识别能力的富含二硫键多肽识别分子。此外，以富含二硫键多肽识别分子为工具，本项目初步开展了细胞靶标蛋白识别和功能调控研究，为多肽识别分子的化学生物学和生物医学应用奠定了基础。在本项目的资助下，共计发表研究论文8篇，获得国家发明专利授权2项；培养硕士研究生6名，博士研究生4名。