Cloning, overexpression, purification, and characterization of a new iron superoxide dismutase fromJatropha curcas
Autor: | Fang Chen, Feng Cai, Shenghua Wang, Chao Ouyang, Shun Gao, Bei Niu |
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Rok vydání: | 2012 |
Předmět: |
Biomedical Engineering
Bioengineering Ethylenediaminetetraacetic acid Biology Molecular cloning medicine.disease_cause Applied Microbiology and Biotechnology law.invention Superoxide dismutase chemistry.chemical_compound Affinity chromatography law Drug Discovery medicine Escherichia coli chemistry.chemical_classification Molecular mass Process Chemistry and Technology General Medicine Enzyme chemistry Biochemistry Recombinant DNA biology.protein Molecular Medicine Biotechnology |
Zdroj: | Biotechnology and Applied Biochemistry. 59:338-345 |
ISSN: | 0885-4513 |
Popis: | A novel iron superoxide dismutase (Fe-SOD) gene from Jatropha curcas was cloned and expressed in Escherichia coli BL21 (DE3). This recombinant enzyme was isolated by a combined procedure involving immobilized metal-ion affinity chromatography and ion-exchange chromatography. The apparent molecular mass of this purified enzyme (designated as JcFe-SOD) was 35 kDa on SDS-PAGE. The full-length complementary DNA sequence of JcFe-SOD contained a 918-bp open reading frame encoding a 305-amino-acid precursor of 34.589 kDa. The result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed that the purified enzyme may own two forms: a dimer and a monomer. The enzyme was relatively stable and showed 54% activity when incubated in 70°C for 60 Min. JcFe-SOD was found to have good pH stability in the pH range of 5.5-9.5 at 25°C over 1 H incubation. The activity of this enzyme was gradually inhibited by increasing concentration of H₂O₂, 2-mercaptoethanol, and ethylenediaminetetraacetic acid. An assay of the atomic absorption spectrum showed the presence of 0.41 atom of Fe in each SOD subunit. |
Databáze: | OpenAIRE |
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