title: Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.
abstract: A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.
authors.affiliation: Department of Biochemistry, College of Life Sciences and Biotechnology, Yonsei University, Seoul 120-749, Korea.
authors.forename: Pravesh
authors.initials: P
authors.lastname: Shrestha
authors.affiliation: Department of Biochemistry, College of Life Sciences and Biotechnology, Yonsei University, Seoul 120-749, Korea.
authors.forename: Ji-Hye
authors.initials: JH
authors.lastname: Yun
authors.affiliation: Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea.
authors.forename: Woo Taek
authors.initials: WT
authors.lastname: Kim
authors.affiliation: Department of Dermatology and Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul 137-040, Korea.
authors.forename: Tae-Yoon
authors.initials: TY
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, College of Life Sciences and Biotechnology, Yonsei University, Seoul 120-749, Korea.
authors.forename: Weontae
authors.initials: W
authors.lastname: Lee
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods*; D004050:Ditiocarb / Q000494:pharmacology; D006801:Humans; D018384:Oxidative Stress; D017433:Protein Structure, Secondary; D061987:Sf9 Cells; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004050:Ditiocarb; C441642:SOD3 protein, human; D013482:Superoxide Dismutase
keywords: LC-MS/MS; extracellular matrix (ECM); human extracellular superoxide (hEC-SOD); reactive oxygen species (ROS); sodiumdiethyldithiocarbamate