title: Cloning, expression, and purification of a Cu/Zn superoxide dismutase from Jatropha curcas.
abstract: We report cDNA cloning, expression, purification, and characterization of a novel Cu/ Zn superoxide dismutase (SOD) from Jatropha curcas leaves. The full-length cDNA of this SOD contained a 496-bp open-reading frame (ORF) encoding 162 amino acid residues. The recombinant plasmid containing the SOD coding sequence was introduced into Escherichia coli, and the SOD was expressed as a fusion protein. The recombinant SOD was purified from a high-density fed-batch culture using a combination of immobilized metal ion affinity chromatography (IMAC) and Sephadex G25 desalting chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis indicated that the recombinant SOD was a monomeric protein with a molecular mass of approximately 16.4 kDa. Isoelectric focusing showed that this SOD was a basic protein with pI values of 7.04, 7.33, 8.62, and 8.77. The activity of the SOD was stable at 70 degrees C for 10 min, and in a broad pH range from 4 to 9. The presence of urea (up to 8 M), guanidinium chloride (up to 6 M), and 2-mercaptoethanol (up to 8 mM) had little effect on the activity. The activity decreased gradually with increasing concentrations of imidazole, hydrogen peroxide, and ethylenediaminetetraacetic acid (EDTA). Atomic absorption spectrometry showed the presence of 0.239 copper and 0.258 zinc atoms, respectively, in the SOD polypeptide.
authors: College of Life Sciences, Sichuan University, 610064 Chengdu, China.
authors: Ou-Yang
authors: OY
authors: Chao
authors: Shun
authors: S
authors: Gao
authors: Feng
authors: F
authors: Cai
authors: Tsair-Wang
authors: TW
authors: Chung
authors: Sheng-Hua
authors: SH
authors: Wang
authors: Fang
authors: F
authors: Chen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D031286:Jatropha / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D013482:Superoxide Dismutase