Evaluating the Potential Role of the Small Metalloprotein, Pyrococcus Furiosus Rubredoxin, as a Targeted Cancer Therapeutic and Imaging Agent

Date of Award


Degree Type


Degree Name

Master of Science in Biomedical Sciences

First Advisor

Francis E. Jenney, Jr. PhD

Second Advisor

Bonnie A Buxton, PhD

Third Advisor

Harold Kominsky, PhD


The new occurrence of all types of cancers has steadily risen over the last forty years. A greater understanding of cancer at the cellular and molecular levels has raised the possibility of antineoplastic drugs that specifically target cancer cells and leave healthy tissues unharmed. The small metalloprotein rubredoxin (Rd), from the hyperthermophile Pyrococcus furiosus (Pf), offers a potential new tool for the treatment of cancers. Rubredoxins are small (~ 6 kDa), non-heme iron proteins commonly found in anaerobic prokaryotes, both bacteria and archaea. Extensive studies have shown that Pf holorubredoxin is stable at 100°C for days, and far longer at room temperature. Additionally, the single iron cofactor can be substituted with a number of transition metals, including nickel, cobalt, and gallium, with little to no observable effect on protein stability. This project seeks to investigate the potential for Rd to serve as a novel delivery system for targeting chemotherapeutic agents specifically to cancer cells. An expression vector containing the gene encoding Pf Rd was modified through a series of molecular cloning techniques such that the E epitope tag (GAPVPYPDPLEPR) was incorporated at a central loop between residues D20 and N21 and at the carboxyl terminus after residue D53 and assayed for binding with anti-E tag antibodies. To determine cell adhesion capacity of Rd, the integrin-binding motif arginine-glycineaspartate (RGD) was also incorporated into the protein structure following residue D20 and was be screened for binding in vitro against a T lymphocyte (Jurkat) cell line. Characterization of Rd mutants revealed that all versions of modified Pf Rd retained absorption spectra very similar to that of the wild-type protein and mass spectroscopy confirmed the predicted mass of the mutants. Thermostability analysis showed that modifications to Pf Rd reduced thermal stability in relation to the size and location of the insert, with D20/N21 loop mutants being less stable than carboxyl terminus mutants. Additionally, both E tag and RGD:integrin affinity screening of Rd mutants demonstrated the epitope binding ability of modified Pf Rd. The results of this study demonstrated that Pf Rd has the potential to serve as a cancer targeted therapeutic and imaging agent.

This document is currently not available here.