Exploring metal binding site tolerance of Pyrococcus furiosus Rubredoxin with GCNGRCD peptide tag
Location
Suwanee, GA
Start Date
7-5-2024 1:00 PM
End Date
7-5-2024 4:00 PM
Description
Introduction: Rubredoxin, a small iron-sulfur protein found in hyperthermophilic archaeon Pyrococcus furiosus, exhibits remarkable tolerance to metal ion substitution while maintaining its structural integrity and function.
Objective: This study delves into the manipulation of rubredoxin's metal binding site to explore its tolerance and functional implications. Rubredoxin was purified from Escherichia coli containing a mutant of hyperthermophilic P. furiosus Rd, with a GCNGRCD tag at the C-terminus.
Methods: Expression and purification involved a series of techniques, including heat treatment, sonication, anion exchange, size exclusion, and hydroxyapatite (HAP) column chromatography, as well as SDS-PAGE gel analysis. The mutant allows for testing the effect of tag location in the protein, positioned at the C-terminus where Rd typically ends with D residue. This protein variant can be compared to the wild type and a mutant form with a different tag at an internal position (after residue D20).
Results: The results showcase successful purification of the Rubredoxin protein with the GCNGRCD tag. The column chromatography results showed the presence of a singular peak, indicating an increased presence of one type of Rubredoxin, and Gel electrophoresis also showed successful insertion of the tag.
Conclusion: By substituting the iron atom with various metal ions, we aim to deepen our understanding of rubredoxin's structural characteristics and extend this knowledge to the purification of other mutant forms of the protein. Rubredoxin's unique characteristics make it a potential candidate for cancer research and targeted treatment strategies. By utilizing its metal binding site tolerance, rubredoxin variants could be engineered to deliver targeted treatment to cancerous cells, thereby enhancing the efficacy and precision of cancer therapies.
Embargo Period
5-23-2024
Exploring metal binding site tolerance of Pyrococcus furiosus Rubredoxin with GCNGRCD peptide tag
Suwanee, GA
Introduction: Rubredoxin, a small iron-sulfur protein found in hyperthermophilic archaeon Pyrococcus furiosus, exhibits remarkable tolerance to metal ion substitution while maintaining its structural integrity and function.
Objective: This study delves into the manipulation of rubredoxin's metal binding site to explore its tolerance and functional implications. Rubredoxin was purified from Escherichia coli containing a mutant of hyperthermophilic P. furiosus Rd, with a GCNGRCD tag at the C-terminus.
Methods: Expression and purification involved a series of techniques, including heat treatment, sonication, anion exchange, size exclusion, and hydroxyapatite (HAP) column chromatography, as well as SDS-PAGE gel analysis. The mutant allows for testing the effect of tag location in the protein, positioned at the C-terminus where Rd typically ends with D residue. This protein variant can be compared to the wild type and a mutant form with a different tag at an internal position (after residue D20).
Results: The results showcase successful purification of the Rubredoxin protein with the GCNGRCD tag. The column chromatography results showed the presence of a singular peak, indicating an increased presence of one type of Rubredoxin, and Gel electrophoresis also showed successful insertion of the tag.
Conclusion: By substituting the iron atom with various metal ions, we aim to deepen our understanding of rubredoxin's structural characteristics and extend this knowledge to the purification of other mutant forms of the protein. Rubredoxin's unique characteristics make it a potential candidate for cancer research and targeted treatment strategies. By utilizing its metal binding site tolerance, rubredoxin variants could be engineered to deliver targeted treatment to cancerous cells, thereby enhancing the efficacy and precision of cancer therapies.