Building a pipeline to generate affinity reagents to phosphothreonine epitopes

抗体在基础研究和临床试验中是非常强大的工具，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 在监测翻译后修饰方面发挥了特别有用的作用，例如 磷酸化苏氨酸残基，其通常调节蛋白质的生化活性， 手机定位尽管绝大多数针对磷酸化表位的商业抗体是 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， 与重组亲和试剂不同。一种基于天然存在的 叉头相关（FHA）结构域结合细胞蛋白中的磷酸苏氨酸，具有 可能成为这种翻译后修饰的高选择性亲和试剂。 将构建噬菌体M13文库，其展示四种不同的FHA结构域， 为了通过亲和选择分离亲和试剂， 与生物医学重要细胞中涉及的五种人类蛋白质相对应的磷酸肽 信号通路工程化的FHA结构域，称为磷酸苏氨酸结合结构域， （pTBD），其已经通过用噬菌体抗体的亲和选择从噬菌体文库分离。 将以两种新的方式验证：a）蛋白质印迹法以合成磷酸肽， 连接到麦芽糖结合蛋白（MBP）的C-末端的磷酸肽和B）结合到 构象折叠的靶蛋白，其在确定的位点携带磷酸苏氨酸。在- pTBD的解离速率、解离速率和解离常数将通过表面等离子体激元测量。 在一个实施方案中，靶蛋白的磷酸化和非磷酸化形式的共振（SPR）可以被用于靶蛋白的磷酸化和非磷酸化形式。到 为了证明pTBD可以选择性地结合它们在复杂混合物中的靶标，我们将掺入E。 大肠杆菌和商业HeLa细胞提取物与一系列浓度的磷酸苏氨酸- 结合的目标和监测定量下拉的目标从复杂的混合物 通过用商业抗靶抗体进行蛋白质印迹。成功完成 拟议的实验将导致开发一个管道，以产生高质量的亲和力， 用于天然蛋白质的基于磷酸苏氨酸的表位的试剂。 -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-