Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.
Department of Immunology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.
Protein Expr Purif. 2021 Aug;184:105891. doi: 10.1016/j.pep.2021.105891. Epub 2021 Apr 22.
Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens.
来自脆弱拟杆菌的免疫球蛋白 A（IgA）蛋白酶是一种能够切割两种亚类 IgA 的酶；相比之下，其他细菌蛋白酶仅能切割 IgA1 蛋白。先前的报告描述了 C. ramosum 自然分泌的蛋白酶对正常人体血清 IgA1 和 IgA2m(1)亚类以及分泌型 IgA（SIgA）的活性。确定了其氨基酸序列，并制备了可切割两种亚类 IgA 的重组蛋白酶。在这里，我们报告了针对纯化的人乳 SIgA 的优化表达、纯化、储存条件和活性测试。分离出的高纯度重组 C. ramosum IgA 蛋白酶几乎完全切割了两种亚类的 SIgA。该蛋白酶在 -20°C 下储存超过 10 个月仍保持活性，且酶活性没有明显损失。纯化的 SIgA 片段适用于研究 SIgA 所有与抗原结合和 Fc 依赖性相关的功能，这些功能参与了对黏膜病原体感染的保护。
