Building a pipeline to generate affinity reagents to phosphothreonine epitopes

建立生产磷酸苏氨酸表位亲和试剂的管道

基本信息

批准号：
10481540
负责人：
BRIAN KENNETH KAY
金额：
$ 22.29万
依托单位：
TANGO BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10481540
关键词：
ARNT gene Affinity Amber Amino Acids Antibodies Bacteriophage M13 Bacteriophages Basic Science Binding Binding Proteins Biochemical Biological Sciences Cell Extracts Cells Client Code Codon Nucleotides Complex Mixtures Contract Services Development Digestion Directed Molecular Evolution Disease Dissociation Engineering Epitopes Escherichia coli Eukaryota FHA Domain Hand Hela Cells Human Libraries Location Measures Medical Research Molecular Biology Techniques Molecular Conformation Monitor Monoclonal Antibodies Oncogenic Patients Peptides Phase Phosphopeptides Phosphorylation Phosphothreonine Play Positioning Attribute Post-Translational Protein Processing Property Protein Engineering Protein Kinase Proteins Randomized Reagent Recombinants Research Support Role Sampling Signal Pathway Signal Transduction Site Small Business Innovation Research Grant Specificity Surface Plasmon Resonance System Techniques Terminator Codon Testing Threonine Tissues Tyrosine United States National Institutes of Health Variant Western Blotting Work assay development base experimental study innovation interest liquid chromatography mass spectrometry maltose-binding protein novel polyclonal antibody protein expression research clinical testing scaffold sortase tool

项目摘要

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-

抗体是基础研究和临床测试中极其强大的工具，因为它们提供在检测复杂混合物中感兴趣的蛋白质时具有精确的特异性和亲和力。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，通常调节蛋白质的生化活性和蜂窝位置。虽然绝大多数针对磷酸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。基于天然存在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，具有有潜力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，显示四个不同的 FHA 结构域，具有不同的功能。识别特性，用于通过亲和选择来分离亲和试剂对应于生物医学重要细胞中涉及的五种人类蛋白质的磷酸肽信号通路。工程化的 FHA 结构域，称为磷酸苏氨酸结合结构域（pTBD），通过亲和力选择从噬菌体文库中分离出来磷酸肽，将以两种新颖的方式进行验证：a）合成的蛋白质印迹 b) 与麦芽糖结合蛋白 (MBP) C 末端连接的磷酸肽和 b) 结合在指定位点携带磷酸苏氨酸的构象折叠靶蛋白。上- pTBD 的速率、解离速率和解离常数将通过表面等离子体测量目标蛋白的磷酸化和非磷酸化形式的共振（SPR）。到为了证明 pTBD 可以选择性地结合复杂混合物中的靶标，我们将掺入大肠杆菌。大肠杆菌和商业 HeLa 细胞提取物，含有一系列浓度的磷酸苏氨酸- 合并目标并监测复杂混合物中目标的定量下拉通过使用商业抗靶标抗体进行蛋白质印迹。圆满完成了拟议的实验将导致开发用于产生高质量亲和力的管道天然蛋白质基于磷酸苏氨酸的表位的试剂。 -1-