Research
In graduate school, I was Dr. Joshua Price’s first graduate student at Brigham Young University, Provo, where I investigated the impact of site-specific PEGylation on protein conformational stability. PEGylation of protein side-chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs; however, nonspecific PEGylation can inadvertently place large PEGs near enzyme active sites or protein-protein binding surfaces, where steric hindrance results in decreased biological activity. Site-specific PEGylation strategies allow researchers to avoid these problematic locations; however, it can be difficult to choose a suitable
PEGylation site. Until recently there were no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal loss to biological activity. We hypothesized that a distinguishing characteristic of optimal vs suboptimal sites is the ability of PEG to enhance the protein conformational stability.
The purpose of my project was to answer the question, “can we develop a structure- or sequence-based criteria for predicting optimal PEGylation sites in peptides and proteins?”
During my post-doctoral training in the laboratory of Dr. Champak Chatterjee at The University of Washington, I have been involved in interrogating the mechanistic role of post-translational modifications (PTM) of the tumor suppressor protein p53. The discrete roles p53 are mediated in part by the ~60 residues in its sequence that are known to undergo post-translational modification. The mis-regulation of p53 PTMs, due to genetic mutations that are observed in ~50% of all human cancers or the inactivation/hyperactivation of PTM writer enzymes, contribute to its oncogenic potential. These PTMs include acetylation, phosphorylation, methylation, sumoylation, and ubiquitylation, of which phosphorylation is one of the most studied. Understanding how single and multi-site PTMs regulate p53 function is critical for understanding cellular pathways that are involved in cancer, which will enable the future development of molecularly targeted therapeutics.
The post-translational modification that I am currently studying is SUMOylation (Small Ubiquitin-like Modifier) of the ε−amine of lysine 386 of the p53 protein hereafter called p53SUMO. To prepare p53SUMO, I relied upon protein semisynthesis, utilizing such methods as expressed protein ligation (EPL), protein trans-splicing (PTS), and Fmoc solid-phase peptide synthesis. For EPL and PTS, I mastered cloning, purifying, and characterizing proteins from E. coli to obtain milligram quantities of desired product. I mastered cloning, purifying, and characterizing proteins from E. coli to obtain milligram quantities of desired product. I have successfully synthesized, purified, and refolded p53SUMO. I have also succeeded in over expressing, purifying, and refolding full-length human wild type p53. I confirmed the activity of the refolded proteins in DNA-binding assays and in vitro transcription assays performed in collaboration with the Roeder Lab at Rockefeller University.