Cloning and expression of the manganese superoxide dismutase gene of Escherichia coli in Lactococcus lactis and Lactobacillus gasseri.
The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site downstream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active.
Roy
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Department of Biochemistry, North Carolina State University, Raleigh 27695-7622.
Klaenhammer
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Hassan
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D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007778:Lactobacillus / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics*; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D010269:Paraquat / Q000494:pharmacology; D010957:Plasmids; D013482:Superoxide Dismutase / Q000235:genetics*
D004269:DNA, Bacterial; D008345:Manganese; D013482:Superoxide Dismutase; D010269:Paraquat; D010100:Oxygen