ISOLATION AND PURIFICATION OF SUBUNIT B OF SHIGA TOXIN

志贺毒素B亚基的分离纯化

• 批准号: 5203360
• 负责人: V POZSGAY
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5203360
• 关键词: Shigella; Vibrio cholerae; affinity chromatography; bacterial antigens; bacterial proteins; bacterial toxins; chemical synthesis; fermentation; genetic strain; glycolipids; immunoconjugates; oligosaccharides; protein purification; receptor binding

The Shiga toxins are multimeric proteins consisting of a single enzymatic subunit (subunit A, mw 32 kDa) and a symmetrically arranged, pentameric subunit B (mw 7.7 kDa). The latter mediates the binding of the holotoxin to cell-surface glycolipids as the first step of its endocytosis. Galabiose is the minimum binding receptor that recognizes the toxin, placed either terminally or internally in glycolipids. The binding is stronger with P blood group determinant trisaccharides, such as the Pk antigen and the P1 antigen. Specific adsorbents of Shiga (Shiga-like) toxins may aid the development of serologic tests for rapid diagnosis of Escherichia coli infections, and receptor-analog oligosaccharides that inhibit the binding of these toxins to their cell-surface receptors can be candidates for therapeutics against such diseases. The non-toxic subunit B may be a candidate as a carrier in the synthesis of polysaccharide-protein conjugates. Based on the reported carbohydrate-specificities of subunit B, we developed a simple and efficient method for the preparative scale isolation of this toxin fragment, using a receptor-analog affinity sorbent. Specifically, we synthesized the P1 antigenic trisaccharide equipped with an anchor at its reducing end. The P1 trisaccharide was covalently attached to a solid support through this anchor. The resulting affinity material was used over twenty cycles to isolate subunit B of Shiga toxin from the fermentation fluid of a non-virulet Vibrio cholera strain that containd the gene for this protein, in a semi-automated fashion.

滋贺毒素是多聚体蛋白质，由单一的酶促降解酶组成。 亚基（亚基A，分子量32 kDa）和对称排列的五聚体 亚基B（分子量7.7 kDa）。 后者介导全毒素的结合 细胞表面糖脂作为其内吞作用的第一步。 半乳糖是识别毒素的最小结合受体， 位于糖脂的末端或内部。 所述结合是 更强的P血型决定簇，如Pk 抗原和P1抗原。 滋贺特异性吸附剂（类志贺） 毒素可能有助于血清学试验的发展，用于快速诊断 大肠杆菌感染和受体类似物寡糖， 抑制这些毒素与其细胞表面受体的结合， 可以作为针对这些疾病的治疗剂的候选物。 无毒 亚基B可以作为合成 多糖-蛋白质缀合物。 基于所报告的 碳水化合物特异性的亚基B，我们开发了一个简单的， 一种制备规模分离该毒素的有效方法 片段，使用受体类似物亲和吸附剂。 我们特别 合成了在其上配备有锚的P1抗原三糖， 减少结束。 将P1三糖共价连接到固体上， 通过这个锚支撑。 使用所得亲和材料 经过二十多个循环，从大肠杆菌中分离出滋贺毒素的B亚基。 一种非小病毒霍乱弧菌菌株的发酵液， 这种蛋白质的基因，以一种半自动的方式。