Purification of Rubredoxin from a mutant strain of Pyrococcus furiosus containing a KNGREG affinity tag
Location
Suwanee, GA
Start Date
7-5-2024 1:00 PM
End Date
7-5-2024 4:00 PM
Description
INTRODUCTION: Cancer continues to be the second leading cause of death in the United States, leading to an increased need to investigate alternative cancer therapies. Conventional treatments like chemotherapy often yield significant side effects including drug resistance, compromised immunity and increased degradation of healthy cells. The ‘NGR’ tag (Asn-Gly-Arg) is a peptide sequence that has been shown to interact with APN/CD13 receptors on some cancer cells, which are commonly used as a target for anti-cancer treatments. Pyrococcus furiosus is an anaerobic, hyperthermophilic archaeon that grows optimally in extreme temperature environments (100°C). The species gives rise to a small iron binding protein called Rubredoxin, which, in vitro, can uptake and integrate varying metals.
OBJECTIVE: Our focus is to investigate the purification of recombinant Fe-binding Rubredoxin protein from Escherichia coli cells with a mutant P. furiosus plasmid containing the NGR tag (KNGREG). The goal is to integrate iron into the molecular structure of the protein and conduct purification.
METHODS: The mutant plasmid was introduced into Escherichia coli via transfection, followed by growth in M9 media. The resulting culture then underwent centrifugation to isolate the protein pellet. This was followed by a series of purification steps including heat treatment, sonication, and column chromatography employing anion exchange, size exclusion and hydroxyapatite columns. The efficacy of the purification was then tested through SDS-PAGE gel and analysis. Furthermore, a comparative analysis was conducted between the mutant, wild-type Rubredoxin and another mutant with a CNGRCG tag at the same location to deduce the possibility of insertion of a mutant NGR tag and binding to APN/CD13.
RESULTS: The results showcase successful purification of the Rubredoxin protein with the mutant tag. The column chromatography results showed the presence of singular peak, which indicated increased presence of one type of Rubredoxin. Gel electrophoresis and comparative analysis also deduced successful insertion of the KNGREG tag.
CONCLUSION: The purification process showed positive results. Further investigation is required to understand the ability and efficacy of the mutant NGR tag binding to APN/CD13 in mammalian cancer lines. Successful binding of the tag to the receptor may allow for more targeted cancer treatments, a decrease in associated side effects, and a healthier lifestyle for cancer patients.
Embargo Period
5-23-2024
Purification of Rubredoxin from a mutant strain of Pyrococcus furiosus containing a KNGREG affinity tag
Suwanee, GA
INTRODUCTION: Cancer continues to be the second leading cause of death in the United States, leading to an increased need to investigate alternative cancer therapies. Conventional treatments like chemotherapy often yield significant side effects including drug resistance, compromised immunity and increased degradation of healthy cells. The ‘NGR’ tag (Asn-Gly-Arg) is a peptide sequence that has been shown to interact with APN/CD13 receptors on some cancer cells, which are commonly used as a target for anti-cancer treatments. Pyrococcus furiosus is an anaerobic, hyperthermophilic archaeon that grows optimally in extreme temperature environments (100°C). The species gives rise to a small iron binding protein called Rubredoxin, which, in vitro, can uptake and integrate varying metals.
OBJECTIVE: Our focus is to investigate the purification of recombinant Fe-binding Rubredoxin protein from Escherichia coli cells with a mutant P. furiosus plasmid containing the NGR tag (KNGREG). The goal is to integrate iron into the molecular structure of the protein and conduct purification.
METHODS: The mutant plasmid was introduced into Escherichia coli via transfection, followed by growth in M9 media. The resulting culture then underwent centrifugation to isolate the protein pellet. This was followed by a series of purification steps including heat treatment, sonication, and column chromatography employing anion exchange, size exclusion and hydroxyapatite columns. The efficacy of the purification was then tested through SDS-PAGE gel and analysis. Furthermore, a comparative analysis was conducted between the mutant, wild-type Rubredoxin and another mutant with a CNGRCG tag at the same location to deduce the possibility of insertion of a mutant NGR tag and binding to APN/CD13.
RESULTS: The results showcase successful purification of the Rubredoxin protein with the mutant tag. The column chromatography results showed the presence of singular peak, which indicated increased presence of one type of Rubredoxin. Gel electrophoresis and comparative analysis also deduced successful insertion of the KNGREG tag.
CONCLUSION: The purification process showed positive results. Further investigation is required to understand the ability and efficacy of the mutant NGR tag binding to APN/CD13 in mammalian cancer lines. Successful binding of the tag to the receptor may allow for more targeted cancer treatments, a decrease in associated side effects, and a healthier lifestyle for cancer patients.