Expression and stability of the nontoxic component of the botulinum toxin complex
Expression and stability of the nontoxic component of the botulinum toxin complex
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DOI：
10.1016/j.bbrc.2009.04.095
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发表时间：
2009-06-19
影响因子：
3.1
通讯作者：
Ohyama, Tohru
中科院分区：
文献类型：
--
作者：
Miyata, Keita;Yoneyama, Tohru;Ohyama, Tohru
肉毒杆菌产生肉毒神经毒素（BoNT）作为一种与无毒非血凝素（NTNHA）和/或血凝素成分相关的大毒素复合物。本研究在大肠杆菌系统中实现了肉毒杆菌血清型D菌株4947 （D-4947）全长（1197个氨基酸）rNTNHA的高水平表达。在蛋白酶抑制剂存在的长期孵育过程中，观察到rNTNHA在特定部位的自发切口；这在天然的NTNHA中也观察到。rNTNHA与分离的D-4947 BoNT以1:1的摩尔比组装形成毒素复合物。尽管分离的BoNT和rNTNHA蛋白都很容易降解，但重组的毒素复合物在高浓度下对胃蛋白酶或胰蛋白酶的蛋白水解表现出明显的抗性。我们提供了明确的证据，证明NTNHA在保护BoNT（一种口服毒素）方面起着至关重要的作用，BoNT是胃和肠中常见的蛋白酶的消化。(C) 2009爱思唯尔公司版权所有。
Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex associated with nontoxic-nonhemagglutinin (NTNHA) and/or hemagglutinin components. In the present Study, high-level expression of full-length (1197 amino acids) rNTNHA from C. botulinum serotype D strain 4947 (D-4947) was achieved in an Escherichia coli system. Spontaneous nicking of the rNTNHA at a specific site was observed during long-term incubation in the presence of protease inhibitors; this was also observed in natural NTNHA. The rNTNHA assembled with isolated D-4947 BoNT with molar ratio 1:1 to form a toxin complex. The reconstituted toxin complex exhibited dramatic resistance to proteolysis by pepsin or trypsin at high concentrations, despite the fact that the isolated BoNT and rNTNHA proteins were both easily degraded. We provide definitive evidence that NTNHA plays a crucial role in protecting BoNT, which is an oral toxin, front digestion by proteases common in the stomach and intestine. (C) 2009 Elsevier Inc. All rights reserved.