Title: "Engineering Synthetic Post-Translational Modifications for Cellular Signaling"
John Ngo, PhD – BME (Advisor, Chair)
Mary Dunlop, PhD – BME
Xue Han, PhD – BME
Wilson Wong, PhD – BME
In addition to the large number of different proteins produced by cells, the diversity of protein function is vastly expanded by how these proteins are modified after translation. Post-translational modifications allow proteins to quickly respond to different inputs with small chemical tags, which can drastically change the function of the protein and alter downstream signaling processes. Despite the prevalence and functionality of post-translational modifications in native biology, existing synthetic biology systems do not efficiently leverage this model. Ideally, synthetic modifications to proteins in engineered cells would be orthogonal to the native biology: the modifying enzyme would be selective for the substrate of interest and proteins that recognize the modification would not bind to any other cellular proteins. For this reason, site-specific enzymatic biotinylation is an attractive choice in the development of an orthogonal post-translational modification. In this work, I will demonstrate the use and functionality of E. coli Biotin Ligase as an enzyme to install post-translational modifications in mammalian cells. I will examine its use in extracellular signaling contexts with cell surface and secreted proteins, as well as in intracellular signaling, where the strategic use of synthetic post-translational modifications can be beneficial in engineered cellular signaling pathways. Finally, I will develop a new orthogonal mode of post-translational modification through the enzymatic addition of adenosine monophosphate and demonstrate its use in signaling and protein-specific cell-surface labeling.