Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
Methods Mol Biol. 2020;2109:251-260. doi: 10.1007/7651_2019_258.
The epithelium forms tight junctions by sealing the paracellular space, and tight junctions prevent the free movement of solutes. Claudin is an important structural and functional component of tight junctions and contributes to the formation of paracellular pathways for different populations of size- and charge-selective solutes. Therefore, modulation of tight junctions is important to develop drug delivery strategies. Clostridium perfringens enterotoxin (CPE) causes food poisoning in humans and is a 35-kDa polypeptide, consisting of 319 amino acids and two functional regions. The C-terminal region of CPE (C-CPE) is not cytotoxic and binds to its receptor claudin, which in turn modulates the epithelial tight junction barrier. Thus, claudin binders, such as C-CPE, are useful tools for drug delivery targeting tight junctions. Here, we provide a protocol for the expression and purification of recombinant C-CPE proteins as claudin binders, an analysis method for C-CPE binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.
上皮细胞通过封闭细胞旁间隙形成紧密连接，而紧密连接阻止溶质的自由移动。Claudin 是紧密连接的重要结构和功能组成部分，有助于形成不同大小和电荷选择性溶质的细胞旁途径。因此，调节紧密连接对于开发药物输送策略很重要。产气荚膜梭菌肠毒素（CPE）会导致人类食物中毒，是一种 35kDa 的多肽，由 319 个氨基酸和两个功能区域组成。CPE 的 C 端区域（C-CPE）没有细胞毒性，与它的受体 Claudin 结合，进而调节上皮细胞紧密连接屏障。因此，Claudin 结合物，如 C-CPE，是针对紧密连接的药物输送的有用工具。在这里，我们提供了一种表达和纯化重组 C-CPE 蛋白作为 Claudin 结合物的方案、一种用于分析 C-CPE 结合亲和力的方法以及一种评估调节紧密连接完整性的效果的程序。
