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dc.identifier.citationXuereb E. (1995). Oxidative stress and expression of superoxide dismutase in escherichia coli and thermus aquaticus (Master's dissertation).en_GB
dc.description.abstractEscherichia coli is a facultative anaerobe and mesophile which produces two types of superoxide dismutase, iron (FeSOD) and manganese (MnSOD), the latter under the control of several global regulators of gene expression. Although much is known about the control ofMnSOD expression, FeSOD has always been assumed to be constitutively expressed. However, it has been suggested that the Fur (ferric uptake regulation) protein may be an inducer of this gene. Conflicting reports in the literature about the oxidative stress response of E.coli which overproduce plasmid-encoded FeSOD where addressed by assessing the types of plasmid used and, in particular, the type of promoter encoded by the plasmid. Strains which overproduced FeSOD unqer the control of the natural (wild-type) promoter were more sensitive to paraquat-induced oxidative stress than were wild type E. coli. Strains which overproduced F eSOD under the control of the artificial trc promoter were less sensitive to oxidative stress. Therefore the FeSOD promoter has a significant influence on the physiological response of the cell to oxidative stress and this result may be suggestive of a further mechanism of regulation. The gene encoding MnSOD from E. coli was cloned and expressed in several different E. coli strains. Tt was found that a manganese supplemented medium was required for the full activity of the enzyme, a result which explains other findings in the literature. In direct contrast, Thermus aquaticus is an obligate aerobe and thermophile which produces only MnSOD and although its gene has been cloned, nothing is known about the response of this important enzyme to oxidative stress in this organism. A bench-top 5 litre fermenter was installed as part of this work and facilitated experiments to investigate oxidative stress in Thermus aquaticus whereby pH and dissolved oxygen levels could be accurately monitored and controlled. A twofold increase in the expression and corresponding activity ofMnSOD was observed when Thermus aquaticus was subjected to oxidative stress using 200 µM paraquat. Surprisingly, no effect was observed when culture conditions included pure oxygen supplied at ambient pressure. A reduction in MnSOD activity was observed when using minimal broth which lacked manganese supplementation, an effect repeated when the iron concentration in the medium was increased. This is suggestive of an in vivo metal substitution in the MnSOD enzyme similar to that described for E. coli MnSOD. The fermentation technology employed to study T. aquaticus was used to investigate conditions for maximum expression and yield of recombinant human MnSOD which may in the near future gain pharmacological relevance. E. coli were used to express this protein under the control of the trc promoter and both maximal expression and cell yield were obtained using a glycerol and phosphate rich broth (terrific broth) Co-overexpression of the E. coli chaperonins GroES and GroEL from a second plasmid resulted in a slight (1.8 fold) increase in the measured specific activity of the SOD enzyme. This contrasts with the 3 fold increase previously observed with human copper-zinc SOD in the presence of these protein chaperones and may reflect a non-specific selectivity of these chaperones for β-sheet proteins (i.e. Copper-zinc SOD) over predominantly a-helical ones (i.e. MnSOD). During investigations on E. coli SOD expressiOn, a previously unreported band of SOD-like activity was observed only AFTER incubation of native polyacrylamide gels with hydrogen peroxide at 5 mM. Further investigation ruled out the possibility of an artefact due to buffers and solutions. Its significance remains enigmatic.en_GB
dc.subjectOxidative stressen_GB
dc.subjectSuperoxide dismutaseen_GB
dc.subjectEscherichia colien_GB
dc.titleOxidative stress and expression of superoxide dismutase in escherichia coli and thermus aquaticusen_GB
dc.rights.holderThe copyright of this work belongs to the author(s)/publisher. The rights of this work are as defined by the appropriate Copyright Legislation or as modified by any successive legislation. Users may access this work and can make use of the information contained in accordance with the Copyright Legislation provided that the author must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the prior permission of the copyright holderen_GB
dc.publisher.institutionUniversity of Maltaen_GB
dc.publisher.departmentFaculty of Medicine & Surgery. Department of Physiology and Biochemistryen_GB
dc.contributor.supervisorHunter, G.J.-
dc.contributor.supervisorHunter, T.-
dc.contributor.creatorXuereb, Edward-
Appears in Collections:Dissertations - FacM&S - 1995
Dissertations - FacM&SPB - 1975-1996

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