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

• 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
• 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

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-

抗体在基础研究和临床测试中是非常强大的工具，因为它们提供