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dc.contributor.authorPortnoff, Alyseen_US
dc.identifier.otherbibid: 8442197
dc.description.abstractThe ubiquitin-proteasome pathway (UPP) is the main route of protein degradation in eukaryotic cells and aids in regulation of cell cycle and cellular homeostasis. This robust pathway can also be utilized for reverse genetics to accelerate the degradation of otherwise stable cellular target proteins. In this work, we present a generalizable approach for protein knockdown by developing chimera proteins, called "ubiquibodies", which combine the activity of an E3 ubiquitin ligase with the affinity of designer binding proteins (DBPs). Specifically, we have utilized the modular E3 ubiquitin ligase CHIP and replaced its natural substrate-binding domain with antibody mimetic binding domains to create various ubiquibodies. Next, we optimized the chimeric construct expression in E. coli and purified uAbs to test their functional activity in vitro. Ubiquibodies were evaluated for both their target binding and subsequent target ubiquitination in vitro. This was further analyzed using mass spectroscopy to determine substrate ubiquitination sites and chain linkages. Within the eukaryotic cellular context, ubiquibodies were tested for their ability to specifically ubiquitinate and degrade their target proteins. Finally, preliminary work was performed using rational design to improve uAb E2 specificity, ensure flexibility for substrate binding and reduce autoubiquitination. From this foundation, we foresee the ubiquibody technology being a powerful tool to enable the dissection of protein function, including post-translational modifications, and the selective degradation of proteins that underlie human disease.en_US
dc.subjectprotein engineeringen_US
dc.subjecttargeted proteolysisen_US
dc.subjectreverse geneticsen_US
dc.titleUbiquibodies: Engineered Ubiquitin Ligases With Unnatural Substrate Specificity For Targeted Protein Silencingen_US
dc.typedissertation or thesisen_US Engineering Universityen_US of Philosophy D., Biomedical Engineering
dc.contributor.chairDelisa, Matthewen_US
dc.contributor.committeeMemberDaniel, Susanen_US
dc.contributor.committeeMemberNicholson, Linda Ken_US

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