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Molecular characterization of a recombinant manganese superoxide dismutase from Lactococcus lactis M4.

Tan BH, Chor Leow T, Foo HL, Abdul Rahim R - Biomed Res Int (2014)

Bottom Line: It was stable up to 45°C.The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD.Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.

ABSTRACT
A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

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Nucleotide sequence and deduced amino acid sequence of SOD gene from pRSET/SOD. The asterisk denotes the stop codon.
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fig1: Nucleotide sequence and deduced amino acid sequence of SOD gene from pRSET/SOD. The asterisk denotes the stop codon.

Mentions: A gene encoding superoxide dismutase (SOD) was amplified from L. lactis M4 on the basis of L. lactis subsp. cremoris MG1363 sodA sequence. The gene was cloned into pCR-Blunt II-TOPO and then subcloned into pRSETA expression vector under the regulation of T7 promoter. Transformants containing the constructed plasmid (pRSET/SOD) were selected on LB agar plates containing appropriate antibiotic. Positive clones were verified by PCR, restriction enzyme digestion analysis, and sequencing. BLASTN analysis of the sequencing result showed 98% identity to the published nucleotide sequence sodA in the genome of L. lactis subsp. lactis IL1403 (accession number AE005176) and 99.5% identity to respective SOD sequence of L. lactis subsp. lactis IL1403. These results showed that the full-length sodA from L. lactis M4 (accession number FJ905108) comprised 621 nucleotides that could encode a protein of 206 amino acids (Figure 1). The only variant of amino acid was found at amino acid 202 of SOD, where it was Tyr and Asp for L. lactis subsp. lactis IL1403 and L. lactis M4, respectively. Expression of the recombinant SOD in E. coli BL21(DE3)pLysS was induced with 1 mM of IPTG. SDS-PAGE of the total expressed protein showed an overexpressed protein band of about 27 kDa in molecular mass (Figure 2(a)), which is in agreement with the combined molecular mass of lactococcal SOD (24 kDa) and 6xHis-tag (3 kDa).


Molecular characterization of a recombinant manganese superoxide dismutase from Lactococcus lactis M4.

Tan BH, Chor Leow T, Foo HL, Abdul Rahim R - Biomed Res Int (2014)

Nucleotide sequence and deduced amino acid sequence of SOD gene from pRSET/SOD. The asterisk denotes the stop codon.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921932&req=5

fig1: Nucleotide sequence and deduced amino acid sequence of SOD gene from pRSET/SOD. The asterisk denotes the stop codon.
Mentions: A gene encoding superoxide dismutase (SOD) was amplified from L. lactis M4 on the basis of L. lactis subsp. cremoris MG1363 sodA sequence. The gene was cloned into pCR-Blunt II-TOPO and then subcloned into pRSETA expression vector under the regulation of T7 promoter. Transformants containing the constructed plasmid (pRSET/SOD) were selected on LB agar plates containing appropriate antibiotic. Positive clones were verified by PCR, restriction enzyme digestion analysis, and sequencing. BLASTN analysis of the sequencing result showed 98% identity to the published nucleotide sequence sodA in the genome of L. lactis subsp. lactis IL1403 (accession number AE005176) and 99.5% identity to respective SOD sequence of L. lactis subsp. lactis IL1403. These results showed that the full-length sodA from L. lactis M4 (accession number FJ905108) comprised 621 nucleotides that could encode a protein of 206 amino acids (Figure 1). The only variant of amino acid was found at amino acid 202 of SOD, where it was Tyr and Asp for L. lactis subsp. lactis IL1403 and L. lactis M4, respectively. Expression of the recombinant SOD in E. coli BL21(DE3)pLysS was induced with 1 mM of IPTG. SDS-PAGE of the total expressed protein showed an overexpressed protein band of about 27 kDa in molecular mass (Figure 2(a)), which is in agreement with the combined molecular mass of lactococcal SOD (24 kDa) and 6xHis-tag (3 kDa).

Bottom Line: It was stable up to 45°C.The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD.Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.

ABSTRACT
A superoxide dismutase (SOD) gene of Lactococcus lactis M4 was cloned and expressed in a prokaryotic system. Sequence analysis revealed an open reading frame of 621 bp which codes for 206 amino acid residues. Expression of sodA under T7 promoter exhibited a specific activity of 4967 U/mg when induced with 1 mM of isopropyl-β-D-thiogalactopyranoside. The recombinant SOD was purified to homogeneity by immobilised metal affinity chromatography and Superose 12 gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses of the recombinant SOD detected a molecular mass of approximately 27 kDa. However, the SOD was in dimer form as revealed by gel filtration chromatography. The purified recombinant enzyme had a pI of 4.5 and exhibited maximal activity at 25°C and pH 7.2. It was stable up to 45°C. The insensitivity of this lactococcal SOD to cyanide and hydrogen peroxide established that it was a MnSOD. Although it has 98% homology to SOD of L. lactis IL1403, this is the first elucidated structure of lactococcal SOD revealing active sites containing the catalytic manganese coordinated by four ligands (H-27, H-82, D-168, and H-172).

Show MeSH