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Cloning, expression and antioxidant activity of a thioredoxin peroxidase from Branchiostoma belcheri tsingtaunese

View Article: PubMed Central - PubMed

ABSTRACT

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that catalyze the thioredoxin- dependent reduction of hydroperoxides. In this study, a novel thioredoxin peroxidase (Bbt-TPx1), a member of the peroxiredoxin superfamily, was found by EST sequence analysis of a cDNA library of Branchiostoma belcheri tsingtaunese ovary. The sequence of a full-length cDNA clone contained an open reading frame encoding a polypeptide of 198 amino acid residues, with a calculated molecular weight of 22,150 Da. The expression patterns of the protein at different developmental stages and adult amphioxus tissues indicate that this enzyme may play important roles in anti-oxidation and innate immunity. The recombinant Bbt-TPx1 protein was expressed with a polyhistidine-tag in Escherichia coli and purified using Ni chromatography followed by SP cation exchange chromatography. The rBbt-TPx1 protein existed as a dimer under non-reducing conditions, and was dissociated into monomers by dithiothreitol (DTT); it might predominantly exist in oligomeric form. The rBbt-TPx1 protein showed a significant thiol-dependent peroxidase activity, removing hydrogen peroxide in the presence of dithiothreitol (DTT), but not glutathione (GSH). Protection of plasmid DNA and the thiol-protein from damage by metal-catalyzed oxidation (MCO) in vitro was also revealed.

No MeSH data available.


Related in: MedlinePlus

Properties of recombinant Bbt-TPx1.(A) SDS-PAGE (12% gel) analysis of rBbt-TPx1 in reducing conditions with Coomassie Brilliant Blue R-250 staining. Lane 1: rBbt-TPx1 purified by Ni2+ chelate column; lane 2: rBbt-TPx1 of lane 1 purified by SP-Sepharose column; lane M: molecular weight markers. (B) SDS-PAGE (12% gel) analysis of reduced and non-reduced rBbt-TPx1 samples. Lane 1: reduced rBbt-TPx1; lane 2: non-reduced rBbt-TPx1; lane M: molecular weight markers. (C) HPLC analysis of rBbt-TPx1 before (a) and after (b) treatment with 50 mM DTT for 12h.
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pone.0175162.g003: Properties of recombinant Bbt-TPx1.(A) SDS-PAGE (12% gel) analysis of rBbt-TPx1 in reducing conditions with Coomassie Brilliant Blue R-250 staining. Lane 1: rBbt-TPx1 purified by Ni2+ chelate column; lane 2: rBbt-TPx1 of lane 1 purified by SP-Sepharose column; lane M: molecular weight markers. (B) SDS-PAGE (12% gel) analysis of reduced and non-reduced rBbt-TPx1 samples. Lane 1: reduced rBbt-TPx1; lane 2: non-reduced rBbt-TPx1; lane M: molecular weight markers. (C) HPLC analysis of rBbt-TPx1 before (a) and after (b) treatment with 50 mM DTT for 12h.

Mentions: The cDNA encoding Bbt-TPx1 was amplified using specific primers. The PCR product was cloned into the pET32a (+) M expression vector and expressed in E. coli as a histidine fusion protein. The rBbt-TPx1 protein was purified by Ni2+ chelate column, resulting in a >80% purity; the protein’s molecular mass was about 24 kDa according to SDS-PAGE (Fig 3A, Lane 1). The rBbt-TPx1 protein was further purified by SP-Sepharose column, and >99% purity was obtained (Fig 3A, Lane 2). The fusion protein showed a slightly larger size compared with the predicted molecular mass of 22.15 kDa, due to the N-terminal leader peptide of 2 kDa encoded by the expression vector.


Cloning, expression and antioxidant activity of a thioredoxin peroxidase from Branchiostoma belcheri tsingtaunese
Properties of recombinant Bbt-TPx1.(A) SDS-PAGE (12% gel) analysis of rBbt-TPx1 in reducing conditions with Coomassie Brilliant Blue R-250 staining. Lane 1: rBbt-TPx1 purified by Ni2+ chelate column; lane 2: rBbt-TPx1 of lane 1 purified by SP-Sepharose column; lane M: molecular weight markers. (B) SDS-PAGE (12% gel) analysis of reduced and non-reduced rBbt-TPx1 samples. Lane 1: reduced rBbt-TPx1; lane 2: non-reduced rBbt-TPx1; lane M: molecular weight markers. (C) HPLC analysis of rBbt-TPx1 before (a) and after (b) treatment with 50 mM DTT for 12h.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383247&req=5

pone.0175162.g003: Properties of recombinant Bbt-TPx1.(A) SDS-PAGE (12% gel) analysis of rBbt-TPx1 in reducing conditions with Coomassie Brilliant Blue R-250 staining. Lane 1: rBbt-TPx1 purified by Ni2+ chelate column; lane 2: rBbt-TPx1 of lane 1 purified by SP-Sepharose column; lane M: molecular weight markers. (B) SDS-PAGE (12% gel) analysis of reduced and non-reduced rBbt-TPx1 samples. Lane 1: reduced rBbt-TPx1; lane 2: non-reduced rBbt-TPx1; lane M: molecular weight markers. (C) HPLC analysis of rBbt-TPx1 before (a) and after (b) treatment with 50 mM DTT for 12h.
Mentions: The cDNA encoding Bbt-TPx1 was amplified using specific primers. The PCR product was cloned into the pET32a (+) M expression vector and expressed in E. coli as a histidine fusion protein. The rBbt-TPx1 protein was purified by Ni2+ chelate column, resulting in a >80% purity; the protein’s molecular mass was about 24 kDa according to SDS-PAGE (Fig 3A, Lane 1). The rBbt-TPx1 protein was further purified by SP-Sepharose column, and >99% purity was obtained (Fig 3A, Lane 2). The fusion protein showed a slightly larger size compared with the predicted molecular mass of 22.15 kDa, due to the N-terminal leader peptide of 2 kDa encoded by the expression vector.

View Article: PubMed Central - PubMed

ABSTRACT

Peroxiredoxins (Prxs) are ubiquitous antioxidant enzymes that catalyze the thioredoxin- dependent reduction of hydroperoxides. In this study, a novel thioredoxin peroxidase (Bbt-TPx1), a member of the peroxiredoxin superfamily, was found by EST sequence analysis of a cDNA library of Branchiostoma belcheri tsingtaunese ovary. The sequence of a full-length cDNA clone contained an open reading frame encoding a polypeptide of 198 amino acid residues, with a calculated molecular weight of 22,150 Da. The expression patterns of the protein at different developmental stages and adult amphioxus tissues indicate that this enzyme may play important roles in anti-oxidation and innate immunity. The recombinant Bbt-TPx1 protein was expressed with a polyhistidine-tag in Escherichia coli and purified using Ni chromatography followed by SP cation exchange chromatography. The rBbt-TPx1 protein existed as a dimer under non-reducing conditions, and was dissociated into monomers by dithiothreitol (DTT); it might predominantly exist in oligomeric form. The rBbt-TPx1 protein showed a significant thiol-dependent peroxidase activity, removing hydrogen peroxide in the presence of dithiothreitol (DTT), but not glutathione (GSH). Protection of plasmid DNA and the thiol-protein from damage by metal-catalyzed oxidation (MCO) in vitro was also revealed.

No MeSH data available.


Related in: MedlinePlus