Date of Award
Master of Science in Biomedical Sciences
Francis E. Jenney, Jr, PhD
Kimberly Deadmond, PhD
Mary P Owen, PhD JD
Cancer is an elusive neoplastic disease that claims the lives of many people around the world every year. Though treatments have become more specific to the different types of cancer, the need remains for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity. The search for a targeted cancer therapeutic is necessary and urgent, and Pyrococcus furiosus rubredoxin might be such a tool. Rubredoxin is a small (53 amino acids), water soluble, non-heme iron electron transfer protein that contains an iron atom cofactor bound by the sulfurs of four cysteine residues, which contribute to the redox activity of this protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project was to incorporate tumor specific
binding sequences at the central loop, express, and purify these recombinant
rubredoxin proteins. Next, the wild-type and recombinant rubredoxins were
characterized based on absorption spectra, thermostability, metal content, and
antibody affinity. Lastly, the effect of wild-type and recombinant rubredoxins
was assessed on cancer cells in-vitro.
The Pyrococcus furiosus rubredoxin gene was manipulated via site-directed mutagenesis to incorporate two test epitopes (E-tag and RGD-tag) at the central loop (between D20 and N21 position). The mutant proteins (D20-Etag and D20-RGD) were purified and analyzed using absorption spectroscopy, thermostability, SDS-PAGE, electrospray ionization mass spectroscopy (ESI-MS), and inductively coupled mass spectrometry (ICP-MS). Binding studies for the D20-Etag mutant were done using a dot blot. Lastly, integrin-stimulated Jurkat cancer cells were incubated with wild-type rubredoxin, D20-Etag, D20-RGD, and the cells were assayed for apoptosis via gel electrophoresis at 24 and 48 hour time points.
The E-tag epitope was successfully incorporated between the D20 and N21 amino acid residues using site directed mutagenesis. The D20-Etag and D20-RGD mutant rubredoxin proteins were successfully expressed, purified, and analyzed. There was an apoptotic effect of D20-RGD rubredoxin on the Jurkat cell line. These results provided a further understanding and appreciation of rubredoxin as a potential targeted therapeutic to cancer cells.
Chirumamilla, Siri Lakshmi, "Characterization of a Hyperthermophilic Redox Protein, Rubredoxin, as a Potential Targeted Cancer Therapeutic" (2012). PCOM Biomedical Studies Student Scholarship. 48.