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Directed evolution of SUMO and Ubiquitin proteases to generate high affinity substrate traps


Post translational modifications such as ubiquitin and the Small Ubiquitin-Like Modifier (SUMO) regulate numerous cellular processes and are evolutionarily conserved in all eukaryotes. Studies on these post translational modifications often require the enrichment of their endogenous conjugates prior to analysis because the modified proteins exist transiently and are low in abundance. Current approaches for isolating these modified proteins contain several unaddressed issues that question the reliability of these approaches. Remarkably, it has been recently demonstrated that the catalytically inactive form of the SUMO protease Ulp1 can be used as a purification tool to purify SUMOylated proteins from yeast cell extracts. In the first chapter of this thesis, we used yeast surface display-based directed evolution to engineer the binding affinity of Ulp1 to further improve the purification strategy. In the second part of this thesis, we identified CthUbp15 as the ubiquitin protease most suited for directed evolution in order to create a similar purification tool for ubiquitin studies.

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