Research Project: Selective Reprogramming of Proteases for Therapeutic applications
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University of Florida Department of Chemical Engineering
PI: Dr. Carl Denard
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I am entering into my third semester in the Denard lab. Some of my responsibilities in lab that I complete for each project include molecular cloing of yeast constructs, molecular cloning analysis ( ex. digestion and FPS sequencing), media buffer preparation , and technical writing.
Project #1 : Develop a yeast surface display system for high-throughput protease interrogations
Proteases are critical enzymes that regulate cellular processes and diversify protein function. However, dysregulated proteases are associated with the onset and progression of many diseases. Improving protease specificity is essential for enhancing the efficacy and safety of protease-based therapeutics across diverse applications, including medicine, industry, and research. The goal of this project is to evolve an orthogonal engineered tobacco etch virus protease with increased specificity for glutamate at P1 under kinetic competition. For this project I my specific responsibilities included the transformation of a tobacco etch virus protease into the DNA of Saccharomyces Cerevisiae.
Project #2 : Isolate and characterize substrate-selective insulin-degrading enzyme (IDE) inhibitors.
Inhibition of the insulin-degrading enzyme (IDE) holds promise as a type 2 diabetes treatment by lowering blood glucose levels. Yet, complete IDE inhibition poses challenges as it also cleaves glucagon- a hormone that opposes insulin's effects. The goal of this project is to develop a high-throughput functional selection to isolate nanobodies (Nbs) specifically inhibiting IDE's cleavage of insulin, not glucagon proteolysis. In this project I have been responsible for performing the polymerase chain reactions and HiFi assembly reactions for the formation of the glucagon and insulin substrate cassettes.
Project #3 : Isolate and characterize pH-dependent Cathepsin B (CatB) inhibitors.
Cathepsin B (CatB) is essential for maintaining cellular homeostasis within the low pH of lysosomes, however, its translocation to the near- neutral extracellular environment in numerous cancers leads to the degradation of extracellular matrix (ECM) components, exacerbating the invasiveness of tumor cells. The goal of this project is to preserve CatB’s critical lysosomal functions while inhibiting its pathogenic activity in the cytosol, by isolating Nbs that specifically inhibit CatB solely within the neutral pH of the extracellular space. These inhibitory Nbs could be used in conjunction with chemotherapeutics that induce cancer cell death. This can be done through a high-throughput functional selection to isolate Nbs that inhibit CatB only at the near-neutral pH of the extracellular space, and not at the lysosomal pH of 4.6. This project will allow for a better understanding of CatB's role in discrete organelle compartments with unique pH levels and facilitates the creation of more effective CatB inhibitors for cancer therapeutics. In this project I am responsible for contributing to troubleshooting CatB purification and the molecular cloning for a new yeast surface display platform.