整合素及其潜在配体的纯化与生化表征研究

整合素是一类重要的细胞表面粘附受体。整合素负责细胞内外的信息交换，通过感受细胞由内向外或者由外向内“双向信号”，整合素被激活，进而招募大量的细胞内蛋白质，介导粘着斑的形成。因此，整合素在癌症发生、血管生成、血栓形成、炎症、骨质疏松症和病毒入侵等过程中发挥重要作用。了解整合素及其配体的结构和功能，有助于推动对于整合素如何通过信号传导，调控粘着斑组装和解聚以及各种生理活动具体分子机制的理解。在发育的不同阶段和不同组织中，整合素以不同的α 亚基和β 亚基组成异源二聚体。在本课题中，我们在 HEK293F 细胞和 High Five 昆虫细胞中表达了多种整合素二聚体 αVβ1、整合素 αVβ3、整合素 αVβ5、整合素 α2β1 和整合素 α5β1 细胞外结构域，并通过镍柱亲和层析和快速液相色谱层析技术纯化得到了整合素异源二聚体。通过进一步优化蛋白表达条件、设计点突变、设计蛋白截短体和插入Acid-Base 多肽等方法，我们最终纯化得到了稳定均一的整合素异源二聚体。同时，我们纯化了骨骼重塑相关因子irisin 和 SARS-CoV-2 受体结合结构域，检测它们与整合素潜在的结合能力。通过透射电镜，我们评估了整合素 α5β1 负染样品的均一性，并初步观察到了整合素 α5β1 的结构特征。该项工作优化了多种整合素的纯化，将为后续解析整合素的结构、了解整合素在调控粘着斑蛋白组装和信号传导过程中的结构和功能提供帮助。

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