Document text
Principal Investigator: BRIAN KENNETH KAY
Organization: TANGO BIOSCIENCES, INC.
Fiscal Year: 2022
Award: $222,933
Funding agency: National Institute of General Medical Sciences
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-
Terms: <ARNT><ARNT gene><ATP-protein phosphotransferase><Affinity><Amber><Amino Acids><Antibodies><Antigenic Determinants><Aryl Hydrocarbon Receptor Nuclear Translocator><Bacteriophage M13><Bacteriophages><Basic Research><Basic Science><Binding><Binding Determinants><Binding Proteins><Biochemical><Biologic Sciences><Biological Sciences><Bioscience><Body Tissues><Cell Body><Cell Communication and Signaling><Cell Extracts><Cell Signaling><Cells><Client><Clinical Evaluation><Clinical Testing><Clinical Treatment Moab><Code><Coding System><Codon><Codon Nucleotides><Coliphage M13><Complex Mixtures><Contract Services><Development><Digestion><Dioxin Receptor, Nuclear Translocator><Directed Molecular Evolution><Disease><Disorder><Dissociation><E coli><E. coli><Engineering><Enterobacteria phage M13><Epitopes><Escherichia coli><Eukaryota><Eukaryote><FHA Domain><Forkhead-Associated Domain><HIF-1 Beta Gene><HIF-1Beta Gene><HIF-1beta><HIF1-Beta Gene><HIF1B><HIF1B Gene><HIF1Beta><HIF1beta Gene><Hand><HeLa><Hela Cells><Human><Hypoxia-Inducible Factor 1 Beta Gene><Hypoxia-Inducible Factor 1, Beta subunit><Immunoblotting><Intracellular Communication and Signaling><Kinase Family Gene><L-Threonine><LC/MS><Libraries><Life Sciences><Ligand Binding Protein><Ligand Binding Protein Gene><Location><M13 Phage><Measures><Medical Research><Modern Man><Molecular Biology Techniques><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Monitor><Monoclonal Antibodies><NIH><National Institutes of Health><Nuclear Translocator Gene Dioxin Receptor><Oncogenic><Patients><Peptides><Phages><Phase><Phosphopeptides><Phosphorylation><Phosphothreonine><Play><Position><Positioning Attribute><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Property><Protein Binding><Protein Engineering><Protein Kinase><Protein Modification><Protein Phosphorylation><Proteins><Randomized><Reagent><Recombinants><Research Support><Role><SBIR><Sampling><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Site><Small Business Innovation Research><Small Business Innovation Research Grant><Specificity><Stop Codon><Surface Plasmon Resonance><System><TANGO><Techniques><Termination Codon><Terminator Codon><Testing><Threonine><Threonine Phosphate><Tissues><Translation Stop Signal><Tyrosine><United States National Institutes of Health><Variant><Variation><Western Blotting><Western Immunoblotting><Work><aminoacid><assay development><bacterial virus><base><biological signal transduction><bound protein><clinical test><conformation><conformational state><developmental><directed evolution><experiment><experimental research><experimental study><genetic protein engineering><glycogen synthase a kinase><hydroxyalkyl protein kinase><innovate><innovation><innovative><interest><liquid chromatography mass spectrometry><mAbs><maltose-binding protein><novel><phosphorylase b kinase kinase><polyclonal antibody><protein blotting><protein design><protein expression><randomisation><randomization><randomly assigned><research clinical testing><scaffold><scaffolding><social role><sortase><srtA gene product><tool>