...
首页> 外文期刊>Applied and Environmental Microbiology >Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.
【24h】

Comparison of toxin overlay and solid-phase binding assays to identify diverse CryIA(c) toxin-binding proteins in Heliothis virescens midgut.

机译:毒素覆盖和固相结合测定法的比较,以鉴定Heliosthis virescens中肠中的各种CryIA(c)毒素结合蛋白。

获取原文
   

获取外文期刊封面封底 >>

       

摘要

The binding proteins, or receptors, for insecticidal Bacillus thuringiensis subsp. kurstaki delta-endotoxins are located in the brush border membranes of susceptible insect midguts. The interaction of one of these toxins, CryIA(c), with proteins isolated from Heliothis virescens larval midguts was investigated. To facilitate the identification of solubilized putative toxin-binding proteins, a solid-phase binding assay was developed and compared with toxin overlay assays. The overlay assays demonstrated that a number of proteins of 170, 140, 120, 90, 75, 60, and 50 kDa bound the radiolabeled CryIA(c) toxin. Anion-exchange fractionation allowed the separation of these proteins into three toxin binding fractions, or pools. Toxin overlay assays demonstrated that although the three pools had distinct protein profiles, similar-size proteins could be detected in these three pools. However, determination of toxin affinity by using the solid-phase binding assay showed that only one of the three pools contained high-affinity binding proteins. The Kd obtained, 0.65 nM, is similar to that of the unsolubilized brush border membrane vesicles. Thus, the solid-phase binding assay in combination with the toxin overlay assay facilitates the identification and purification of high-affinity B. thuringiensis toxin-binding proteins from the insect midgut.
机译:杀虫苏云金芽孢杆菌亚种的结合蛋白或受体。库尔斯塔基δ-内毒素位于易感昆虫中肠的刷状缘膜中。研究了这些毒素之一CryIA(c)与从Heliothis virescens幼虫中肠分离的蛋白质的相互作用。为便于鉴定可溶的假定毒素结合蛋白,开发了一种固相结合测定并将其与毒素覆盖测定进行比较。重叠测定表明,许多170、140、120、90、75、60和50 kDa的蛋白质结合了放射性标记的CryIA(c)毒素。阴离子交换分离可将这些蛋白质分离为三个毒素结合部分或库。毒素覆盖分析表明,尽管这三个库具有不同的蛋白质谱,但在这三个库中可以检测到大小相似的蛋白质。但是,通过使用固相结合测定法确定毒素亲和力表明,三个库中只有一个包含高亲和力结合蛋白。所获得的Kd为0.65nM,与未溶解的刷状缘膜囊泡的Kd相似。因此,固相结合测定法与毒素覆盖测定法相结合有助于从昆虫中肠鉴定和纯化高亲和力的苏云金芽孢杆菌毒素结合蛋白。

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