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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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SDS-PAGE analysis of the pooled fractions after each purification step. SDS-PAGE was performed on 12% denatured polyacrylamide gel. (a) SDS-PAGE analysis of pooled fractions with SOD activity after IMAC: M, protein molecular weight marker (Fermentas); lane 1, pooled peak 1 (6 μg); lane 2, pooled peak 2 (6 μg); (b) SDS-PAGE analysis of pooled fractions after gel filtration: M, protein molecular weight marker (Fermentas); lane 1, crude extract (10 μg); lane 2: pooled SOD active fractions (peak 2) after IMAC (4 μg); lane 3: pooled SOD active fractions after gel filtration chromatography (2 μg); lane 4: dialyzed pooled SOD active fractions after gel filtration chromatography (2 μg); (c) SOD activity staining of the purified native SOD electrophoresed on 10% nondenatured polyacrylamide gel. Lane 1: IMAC; (2) gel filtration; (3) dialyzed sample. The achromatic zone against the purple background revealed the activity of SOD. Each lane is loaded with 2 μg of protein.
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fig3: SDS-PAGE analysis of the pooled fractions after each purification step. SDS-PAGE was performed on 12% denatured polyacrylamide gel. (a) SDS-PAGE analysis of pooled fractions with SOD activity after IMAC: M, protein molecular weight marker (Fermentas); lane 1, pooled peak 1 (6 μg); lane 2, pooled peak 2 (6 μg); (b) SDS-PAGE analysis of pooled fractions after gel filtration: M, protein molecular weight marker (Fermentas); lane 1, crude extract (10 μg); lane 2: pooled SOD active fractions (peak 2) after IMAC (4 μg); lane 3: pooled SOD active fractions after gel filtration chromatography (2 μg); lane 4: dialyzed pooled SOD active fractions after gel filtration chromatography (2 μg); (c) SOD activity staining of the purified native SOD electrophoresed on 10% nondenatured polyacrylamide gel. Lane 1: IMAC; (2) gel filtration; (3) dialyzed sample. The achromatic zone against the purple background revealed the activity of SOD. Each lane is loaded with 2 μg of protein.

Mentions: The purification procedure of recombinant SOD is summarized in Table 1. The purity and the estimated molecular weight of recombinant SOD were analyzed with SDS-PAGE after each purification step (Figure 3). The dialyzed recombinant SOD was purified to apparent homogeneity by IMAC and gel filtration chromatography having a purification fold and yield of 3.74 and 22.84%, respectively. The specific activity of purified enzyme was 1.865 × 104 units/mg protein. SOD activity of purified enzyme was visualized on native polyacrylamide gel (PAG) by NBT activity staining. The negatively stained bands against the purple color background of the PAG indicate the activity of SOD (Figure 3(c)).


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)

SDS-PAGE analysis of the pooled fractions after each purification step. SDS-PAGE was performed on 12% denatured polyacrylamide gel. (a) SDS-PAGE analysis of pooled fractions with SOD activity after IMAC: M, protein molecular weight marker (Fermentas); lane 1, pooled peak 1 (6 μg); lane 2, pooled peak 2 (6 μg); (b) SDS-PAGE analysis of pooled fractions after gel filtration: M, protein molecular weight marker (Fermentas); lane 1, crude extract (10 μg); lane 2: pooled SOD active fractions (peak 2) after IMAC (4 μg); lane 3: pooled SOD active fractions after gel filtration chromatography (2 μg); lane 4: dialyzed pooled SOD active fractions after gel filtration chromatography (2 μg); (c) SOD activity staining of the purified native SOD electrophoresed on 10% nondenatured polyacrylamide gel. Lane 1: IMAC; (2) gel filtration; (3) dialyzed sample. The achromatic zone against the purple background revealed the activity of SOD. Each lane is loaded with 2 μg of protein.
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fig3: SDS-PAGE analysis of the pooled fractions after each purification step. SDS-PAGE was performed on 12% denatured polyacrylamide gel. (a) SDS-PAGE analysis of pooled fractions with SOD activity after IMAC: M, protein molecular weight marker (Fermentas); lane 1, pooled peak 1 (6 μg); lane 2, pooled peak 2 (6 μg); (b) SDS-PAGE analysis of pooled fractions after gel filtration: M, protein molecular weight marker (Fermentas); lane 1, crude extract (10 μg); lane 2: pooled SOD active fractions (peak 2) after IMAC (4 μg); lane 3: pooled SOD active fractions after gel filtration chromatography (2 μg); lane 4: dialyzed pooled SOD active fractions after gel filtration chromatography (2 μg); (c) SOD activity staining of the purified native SOD electrophoresed on 10% nondenatured polyacrylamide gel. Lane 1: IMAC; (2) gel filtration; (3) dialyzed sample. The achromatic zone against the purple background revealed the activity of SOD. Each lane is loaded with 2 μg of protein.
Mentions: The purification procedure of recombinant SOD is summarized in Table 1. The purity and the estimated molecular weight of recombinant SOD were analyzed with SDS-PAGE after each purification step (Figure 3). The dialyzed recombinant SOD was purified to apparent homogeneity by IMAC and gel filtration chromatography having a purification fold and yield of 3.74 and 22.84%, respectively. The specific activity of purified enzyme was 1.865 × 104 units/mg protein. SOD activity of purified enzyme was visualized on native polyacrylamide gel (PAG) by NBT activity staining. The negatively stained bands against the purple color background of the PAG indicate the activity of SOD (Figure 3(c)).

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