...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Infection and immunity >Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.
【24h】

Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.

机译：通过在大肠杆菌中的重组表达排除化脓性链球菌促红细胞生成素A制剂中的生物活性污染物。

获取原文
   

获取外文期刊封面封底 >>

       
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.
机译：链球菌的促红细胞外毒素A（SPEA）属于细菌超抗原家族，与中毒性休克样综合征和猩红热的发病机理有关。关于其生物学活性，主要是T细胞刺激特性，存在矛盾的数据。在这项研究中，我们表明迄今为止使用的大多数SPEA制剂均含有生物活性污染物。来自化脓链球菌NY-5的培养上清液中的天然SPEA和从血链球菌培养滤液中纯化的重组SPEA被DNase严重污染。我们显示天然SPEA比重组SPEA诱导更多的肿瘤坏死因子α（TNF-alpha），但我们也显示DNases能够诱导TNF-alpha。在商业SPEA制剂中，我们鉴定出一种高活性蛋白酶，该蛋白酶被证明不是SPEB。为了排除这些污染，我们在大肠杆菌中过表达了有效高效表达载体pIN-III-ompA2中克隆的SPEA。表达的SPEA具有与天然SPEA相同的氨基酸组成，而迄今为止报道的功能研究是针对含有错误氨基末端的毒素进行的。我们描述了快速纯化脂多糖，DNase和无蛋白酶的SPEA从宿主周质中的两个步骤，并通过圆二色性对其结构进行了表征。我们的结果首次代表了在大肠杆菌中生产具有真实N端序列和经验证的超抗原活性的重组SPEA。总的来说，我们的结果表明，超抗原的免疫学研究需要没有生物活性污染物的高纯度物质。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号