# Building a pipeline to generate affinity reagents to phosphothreonine epitopes

> **NIH NIH R43** · TANGO BIOSCIENCES, INC. · 2022 · $222,933

## Abstract

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.
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## Key facts

- **NIH application ID:** 10481540
- **Project number:** 1R43GM146514-01
- **Recipient organization:** TANGO BIOSCIENCES, INC.
- **Principal Investigator:** BRIAN KENNETH KAY
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $222,933
- **Award type:** 1
- **Project period:** 2022-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10481540

## Citation

> US National Institutes of Health, RePORTER application 10481540, Building a pipeline to generate affinity reagents to phosphothreonine epitopes (1R43GM146514-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10481540. Licensed CC0.

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