Dempsey, DanielMerkler, David J.Pi, Kessinee2025-10-212024https://hdl.handle.net/2144/515282024BACKGROUND: Post-translational modifications (PTMs) are processing events that can be added to various proteins following translation. PTMs span a broad spectrum ranging from acetylation, phosphorylation, to ubiquitination and are responsible for various protein-protein interactions and the regulation of many cellular processes. USP7 is a deubiquitinase that has been discovered to play a regulating role on the cancer relevant MDM2-P53 pathway, which makes it an attractive target in developing novel therapeutics for the treatment of cancer. The aim of this present study is to generate post-translationally modified peptides and attach them to truncated versions of USP7 protein expressed in insect cells or E. coli in order to develop a full length semi-synthetic protein with site-specific PTMs. METHODS: Peptide synthesis was done using standard Fmoc strategies and solid phase peptide synthesis techniques. The peptides were purified using reverse phase HPLC and confirmed using MALDI-TOF-MS. Truncated versions of USP7 N∆26 cultured in E. coli were purified using nickel affinity chromatography and ligated to the protein using expressed protein ligation (EPL). Further purification was done using FPLC techniques. SDS-PAGE and fluorescence imaging was used to confirm the presence of our product. CONCLUSIONS: We synthesized four USP7 peptides (Lys1096 acetylated and unacetylated, Ser18 phosphorylated and unphosphorylated) and validated their purity by MALDI-TOF-MS. We also used expressed protein ligation to generate USP7 that is phosphorylated and unphosphorylated at Ser18. The next step would be to characterize the enzymatic properties of USP7 to understand how these PTMs impact its enzymatic function.en-USAttribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/BiochemistryThesis/Dissertation2025-10-21