Angiogenin (ANG) is a secreted protein that holds important implications in the growth and survival for a variety of cell types—from healthy endothelial or neuronal cells, to diseased cancer cells. Currently, we lack a complete understanding of ANG’s mechanism of action. Our work aims to develop a functional fluorescence-based reporting system to study ANG internalization activity in cancer cells. Specifically, we utilize a split green fluorescent protein (split GFP) system, in which the 11th strand of the GFP molecule (GFP11) is attached to ANG, while the remaining non-fluorescent GFP1-10 fragment is expressed in our target HeLaGFP1-10 cell line. The reconstitution of GFP1-10 and GFP11 will restore bright fluorescence signal, indicating ANG’s successful cellular entry. The workflow of this project is two-fold: first, to design, express and purify six variants of ANG-GFP11 proteins, and second, to characterize the kinetics and internalization activity of these fusion proteins in HeLaGFP1-10 cells. We have successfully produced three C-terminal ANG-GFP11 variants with robust ribonucleolytic activities, and demonstrated their capacity to reconstitute GFP fluorescence. With these key tools in place, we are now assessing the system’s efficiency in monitoring ANG uptake. If successful, this split fluorescence system offers a powerful way to study ANG activity in the target cell line, ultimately shedding more light on the role of ANG in cancer and other disease-related pathways.
Level of Honors
summa cum laude
Lê, Phan Quỳnh Châu, "Development of a Split Green Fluorescent Protein Reporting System for Angiogenin" (2021). Lawrence University Honors Projects. 162.
Available for download on Wednesday, June 15, 2022