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Characterization of hyperthermophilic redox protein rubredoxin as a potential targeted cancer therapeutic
Research Day
  • Siri L Chirumamilla, Philadelphia College of Osteopathic Medicine
  • David L. Blum, University of Georgia, Athens, GA
  • Ray L. Mernaugh, Vanderbilt University School of Medicine
  • Francis E. Jenney, Philadelphia College of Osteopathic Medicine
Location
Georgia Campus
Start Date
1-5-2013 2:00 PM
End Date
1-5-2013 4:00 PM
Disciplines
Description

Background: 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 for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity remains, and 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, which can be substituted with various cytotoxic transition metals such as nickel and cobalt with little or no effect on the protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project is to incorporate tumor-specific binding sequences at several modifiable sites on the protein as well as substitute the iron-center with cytotoxic metals. Once a stable rubredoxin containing these characteristics is created, its effects and efficacy will be studied on specific cancer cells in vitro. Methods and results: Site-directed mutagenesis was used to incorporate a test epitope (E-tag) at the D20 position within rubredoxin. An RGD-sequence was previously cloned at the D20 position within rubredoxin. The mutant proteins were purified using anion-exchange DEAE, sizeexclusion G-75 Sephadex, and ceramic hydroxyapatite column chromatography. Proteins were analyzed using absorption spectroscopy, bicinchoninic acid (BCA) assay, SDS-PAGE, electrospray ionization mass spectroscopy, and thermostability. Binding studies for the D20-Etag mutant were done using dot blot. The metal content of the mutants was assessed using inductively-coupled plasma mass spectrometry. Lastly, integrin-stimulated Jurkat cancer cell lines were incubated with wild-type rubredoxin, D20-Etag, D20-RGD, and the effect of these mutants were assessed for apoptosis and necrosis at the 24 hour and 48 hour time points via gel electrophoresis. Conclusions: The epitope E-tag 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 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 for antineoplastic drugs that target cancer cells and leave normal cells unharmed, with little to no systemic toxicity remains, and 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, which can be substituted with various cytotoxic transition metals such as nickel and cobalt with little or no effect on the protein. Rubredoxin from the hyperthermophile Pyrococcus furiosus is thermostable and appears to have low immunogenicity. The focus of this project is to incorporate tumor-specific binding sequences at several modifiable sites on the protein as well as substitute the iron-center with cytotoxic metals. Once a stable rubredoxin containing these characteristics is created, its effects and efficacy will be studied on specific cancer cells in vitro.

Methods and results: Site-directed mutagenesis was used to incorporate a test epitope (E-tag) at the D20 position within rubredoxin. An RGD-sequence was previously cloned at the D20 position within rubredoxin. The mutant proteins were purified using anion-exchange DEAE, sizeexclusion G-75 Sephadex, and ceramic hydroxyapatite column chromatography. Proteins were analyzed using absorption spectroscopy, bicinchoninic acid (BCA) assay, SDS-PAGE, electrospray ionization mass spectroscopy, and thermostability. Binding studies for the D20-Etag mutant were done using dot blot. The metal content of the mutants was assessed using inductively-coupled plasma mass spectrometry. Lastly, integrin-stimulated Jurkat cancer cell lines were incubated with wild-type rubredoxin, D20-Etag, D20-RGD, and the effect of these mutants were assessed for apoptosis and necrosis at the 24 hour and 48 hour time points via gel electrophoresis.

Conclusions: The epitope E-tag 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.

Citation Information
Siri L Chirumamilla, David L. Blum, Ray L. Mernaugh and Francis E. Jenney. "Characterization of hyperthermophilic redox protein rubredoxin as a potential targeted cancer therapeutic" (2013)
Available at: http://works.bepress.com/francis_jenney/7/