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dc.contributor.authorJames, Paul Brian Charlesen_GB
dc.date.accessioned2011-06-29T14:08:41Zen_GB
dc.date.accessioned2013-03-21T10:47:25Z
dc.date.issued2010-09-30en_GB
dc.description.abstractPeroxiredoxins are a family of multifunctional enzymes that are able to protect the cell against oxidative stress. Peroxiredoxins form part of a recently discovered peroxide scavenging system along with thioredoxin, thioredoxin reductase and sulfiredoxin. This study describes the purification of a recombinant human peroxiredoxin II from human erythrocytes. The original recombinant clone contained a point mutation at the fourth residue from glycine to valine and a number of problems were encountered with aggregation during purification. Reverting back to the original amino acid sequence allowed the protein to be purified and concentrated without aggregation, as well as leading to over-expression in the same oligomeric state as the native sample from blood. This study also describes the over-expression and purification of the human peroxiredoxin II protein in the intermolecular disulfide form as well as the subsequent crystallisation and X-ray diffraction studies. The crystal structure for this form of the protein was obtained to 3.3 Å resolution revealing the peroxiredoxin to be in the decameric form. In addition conformational changes in the protein that are necessary for formation of the intermolecular disulfide between the peroxidatic (Cys52) and resolving cysteine (Cys172) have been observed. The structure also revealed that these movements did not interfere with the dimer:dimer interface as had been previously suggested. This then allows the disulfide to be seen within the decameric form of peroxiredoxin. The production of covalent complexes formed between peroxiredoxin and sulfiredoxin, and peroxiredoxin and thioredoxin was also investigated. Complexes were stabilised by using DTNB to form a covalent bond between specific cysteine residues. The complex binding results from size exclusion chromatography showed that decameric peroxiredoxin bound to sulfiredoxin in a 1:5 ratio and decameric peroxiredoxin bound to thioredoxin in a 1:10 ratio. Cloning, over-expression and purification of the selenocysteine containing enzyme thioredoxin reductase was achieved. A minimal selenocysteine insertion sequence was added to the 3’ end of the DNA sequence to drive selenocysteine insertion in place of the typical stop UGA codon. The activity of this protein was found to be low but was greatly increased when co-expressed with a plasmid containing the selA, selB and selC genes. Although the activity of this co-expressed thioredoxin reductase was ~20% of the native enzyme activity, it was comparable to the activity of other recombinant forms of the enzyme. These studies report the purification of all of the proteins necessary to reform the peroxiredoxin system and allow the production of a working assay for peroxiredoxin activity. Together with the first report for a structure of a decameric disulfide form of human peroxiredoxin II a greater insight into the peroxiredoxin system has been obtained.en_GB
dc.identifier.urihttp://hdl.handle.net/10036/3162en_GB
dc.language.isoenen_GB
dc.publisherUniversity of Exeteren_GB
dc.subjectPeroxiredoxinen_GB
dc.subjectThioredoxinen_GB
dc.subjectThioredoxin Reductaseen_GB
dc.subjecthPrxIIen_GB
dc.titleInvestigation into Peroxiredoxin and interactions in the Peroxiredoxin peroxide scavenging systemen_GB
dc.typeThesis or dissertationen_GB
dc.date.available2011-06-29T14:08:41Zen_GB
dc.date.available2013-03-21T10:47:25Z
dc.contributor.advisorLittlechild, Jenniferen_GB
dc.publisher.departmentBiosciencesen_GB
dc.type.degreetitlePhD in Biological Sciencesen_GB
dc.type.qualificationlevelDoctoralen_GB
dc.type.qualificationnamePhDen_GB


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