Building a pipeline to generate affinity reagents to phosphothreonine epitopes

抗体在基础研究和临床测试中是极其强大的工具，因为它们提供了 在检测复杂混合物中感兴趣的蛋白质方面具有极高的特异性和亲和力。他们有 在监控翻译后修改方面发挥了特别有用的作用，例如 磷酸化苏氨酸通常调节蛋白质的生化活性和 蜂窝位置。而绝大多数商用的抗磷表位抗体是 多克隆和单克隆抗体，它们在可再生性和蛋白质工程方面受到限制， 不同于重组亲和试剂。一种基于自然生成的重组支架 叉头相关(FHA)结构域与细胞蛋白中的磷酸苏氨酸结合，具有 有可能成为这种翻译后修饰的高度选择性亲和剂。 将建立噬菌体M13文库，展示四个不同的FHA结构域 用于通过亲和选择分离亲和试剂的识别特性 与生物医学重要细胞相关的五种人类蛋白对应的磷酸肽 信号通路。工程化FHA结构域，称为磷酸苏氨酸结合结构域 (PTbD)，通过亲和选择从噬菌体文库中分离出来。 磷酸肽将以两种新的方式进行验证：a)蛋白质印迹到人工合成 连接到麦芽糖结合蛋白(MBP)C末端的磷酸肽和b)结合 构象折叠的靶蛋白，在特定的位置携带磷苏氨酸。就在- PTBD的速率、离散率和离解常数将通过表面等离子体激元来测量 目标蛋白的磷酸化和非磷酸化形式的共振(SPR)。至 证明pTbd可以选择性地结合它们的目标在复杂的混合物中，我们将尖峰E。 具有一定浓度范围的磷酸苏氨酸的大肠杆菌和商业HeLa细胞提取物- 结合靶并监控复杂混合物中靶的定量下拉 用商品化的抗靶抗体进行免疫印迹。圆满完成 拟议的实验将导致开发一种产生高质量亲和力的管道 天然蛋白质基于磷酸苏氨酸表位的试剂。 -1-

Antibodies are incredibly powerful tools in basic research and clinical testing because they offer exquisite specificity and affinity in detecting proteins of interest in complex mixtures. They have played a particularly useful role in monitoring post-translational modifications, such as phosphorylated threonine resides, which often regulate a protein's biochemical activity and cellular location. While the vast majority of commercial antibodies to phosphoepitopes are polyclonal and monoclonal antibodies, they are limited in renewability and protein engineering, unlike recombinant affinity reagents. A recombinant scaffold based on the naturally occurring Forkhead associated (FHA) domain, which binds phosphothreonines in cellular proteins, has the potential to be a highly selective affinity reagent for this post-translational modification. Bacteriophage M13 libraries will be built that display four different FHA domains with different recognition properties for the purpose of isolating affinity reagents through affinity selection with phosphopeptides corresponding to five human proteins involved in biomedically important cell signalling pathways. The engineered FHA domains, termed phosphothreonine-biding domains (pTBDs), which have been isolated from the phage libraries through affinity selection with the phosphopeptides, will be validated in two novel manners: a) western blotting to synthetic phosphopeptides ligated to the C-terminus of maltose binding protein (MBP) and b) binding to conformationally-folded target proteins that carry phosphothreonine at defined sites. The on- rates, off-rates, and dissociation constants of the pTBDs will be measured by surface plasmon resonance (SPR) for phosphorylated and non-phosphorylated forms of the target proteins. To demonstrate the pTBDs can selectively bind their targets in complex mixtures, we will spike E. coli and commercial HeLa cell extracts with a range of concentrations of the phosphothreonine- incorporated targets and monitor the quantitative pull-down of the targets from complex mixtures by western blotting with commercial anti-target antibodies. Successful completion of the proposed experiments will lead to development of a pipeline for generating high-quality affinity reagents to phosphothreonine-based epitopes of native proteins. -1-