Characterization and Self-Encapsulation of Supercharged Green Fluorescent Proteins by Thermophilic Ferritin

Students

2020
Engineering and Applied Sciences

Faculty

Professor of Chemistry

Project Summary

This summer, I conducted research in Dr. Dmochowski’s biochemistry-oriented lab, studying the characterization and applications of protein-protein interactions. The primary protein in question was thermophilic ferritin from Archaeoglobus fulgidus(AfFtn) , which oxidizes and stores iron in nearly all organisms. In low salt solution, it remains in its disassembled state as a dimer, but in high salt solution the protein forms a hollow cage. We were interested in what proteins or nanoparticles could fit inside that cage, and what properties that entire complex would have.

 

The first protein we decided to encapsulate was supercharged Green Fluorescent Protein(GFP(+36)) because it readily fit into the cage due to electrostatic interactions, and it allowed us to detect proof of encapsulation using a fluorescence microplate reader. We also carried out experiments using a mutant of AfFtn called E65R. This ferritin was unique in that it formed a cage in solutions of varying salt concentrations, so the properties of an E65R-GFP complex were worth studying as well.

 

Although I have had previous research experiences, I have never worked in a biochemistry lab. I therefore learned a considerable amount in a span of ten weeks, ranging from actual research content to lab techniques. The research papers I had to read to familiarize myself with the project strengthened my paper comprehension skills, as well as my ability to search for certain papers online.

The core lab techniques I was taught revolved around protein expression and purification. I learned the entire process of obtaining pure protein from a plasmid, from expressing it in bacteria, to making cultures and purifying the protein using Fast Protein Liquid Chromatography(FPLC). The variety of experiments we ran allowed me to become familiar with several instruments, including a UV-Vis Spectrophotometer, CD Spectrophotometer, sonicator, and tabletop centrifuge. Though not difficult to learn, these procedures and techniques have given me a solid foundation for working in similar labs in the future.

 

I am not a biochemistry major, but I enjoyed working on this project because it gave me the chance to explore something different. Obtaining knowledge from lectures and textbooks is definitely valuable, but when given the opportunity to apply that knowledge, it gives you a sense of purpose and accomplishment. Research such as this not only complements education in the classroom, it enhances education because you begin to understand the “why,” rather than getting stuck on the “what” and the “how.” This summer has been a valuable experience, and will only help me in my future endeavors at Penn.