Advanced Science Research Center, GC/CUNY’s Post

Last week, I had the amazing opportunity to present my research at The City College Academy of Professional Preparation (CCAPP) Annual Poster Presentation. My poster, "Exploring the Role of Highly Networked Residues in PTP1B Activity Using Site-Directed Mutagenesis", talked about the family of protein tyrosine phosphatases and how a specific protein in this family, PTP1B is of interest due to its negative regulation of insulin and leptin signaling which affects glucose metabolism and energy balance. Studies have also shown that overexpression of PTP1B is linked to metabolic disorders such as type 2 diabetes and obesity. More recently, it has been shown that PTP1B is correlated with certain cancers, where it promotes tumor growth and metastasis. I am to figure out what would happen if I mutate highly connected and networked residues within PTP1B using the technique of site-directed mutagenesis. The methods to answer this question included: • Created primers and ran a polymerase chain reaction (PCR) to mutate the selected residues of interest that have varying levels of conservation among the PTP family. • Transformed the PCR products with bacteria cells. • Miniprepped the bacterial plasmid, to obtain a purified plasmid. • Sent samples of the purified plasmid to get sanger sequenced to verify if the mutation took place. • Since, the mutation took place at the desired amino acid, I continued with protein expression. • Verified if the protein expression was successfully done by running an SDS-PAGE gel. Currently, the project is still being conducted and the answer to my question is still being figured out. The next steps would be to conduct protein purification and when the purified protein is obtained, I would perform enzyme kinetic assays and create Michaelis-Menten plots to assess how each mutant affects the functionality of PTP1B. This project is currently being conducted at The Keedy Lab located at the Advanced Science Research Center, GC/CUNY, under the guidance of Dr. Daniel Keedy and mentors Alexis Lemberikman and Shivani Sharma. This has been an incredible journey and I have truly been enjoying my time at Keedy lab. I am excited to continue working on this project and figure out the key findings of this experiment. #research #undergraduateresearch #ASRC #CCNY #CCAPP #PosterPresentation #Proteins