Abstract
Antibodies play a crucial role in life science research for monitoring post-translational modifications, such as phosphorylation, which regulates protein function and cellular behavior. However, current commercial poly- and monoclonal antibodies sometimes suffer from a lack of specificity and variability due to limitations with animal immunization that slows down progress and increases costs. The use of phage display, where billions of protein sequences are presented on a viral coat protein, enables the rapid discovery of highly specific affinity reagents and represents a recombinant approach that eliminates batch variability. In this study, we utilized phage display to screen for affinity reagents that exhibit unparalleled specificity for phosphorylated epitopes, using phosphothreonine as a proof-of-concept. Our library presented variations of a phosphobody scaffold that were screened against the DNA repair protein 53BP1, which features a phosphothreonine at position 543 to regulate its function. The screen identified several hits that were validated in ELISA and Western blot experiments. The affinity reagents could distinguish between phosphorylated vs. unphosphorylated threonine, as well as differentiate phosphorylated threonine vs. phosphorylated serine, demonstrating their ability to identify targets that differ by a single methyl group. These results highlight how phage display screening can identify and optimize highly specific affinity reagents for proteins decorated with phosphorylated residues. This approach can be expanded to other post-translational modifications, thus offering a new toolbox for proteomics and diagnostic research.
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