Cloning, purification, and characterization of recombinant human extracellular superoxide dismutase in SF9 insect cells

Pravesh Shrestha, Ji Hye Yun, Woo Taek Kim, Tae Yoon Kim, Weon Tae Lee

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 (ECSOD), 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.

Original languageEnglish
Pages (from-to)242-249
Number of pages8
JournalMolecules and cells
Volume39
Issue number3
DOIs
Publication statusPublished - 2016 Jan 1

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Superoxide Dismutase
Insects
Organism Cloning
Oxidative Stress
Reactive Oxygen Species
Ditiocarb
Baculoviridae
Superoxides
Extracellular Matrix
Plasmids
Homeostasis
Antioxidants
Western Blotting
Lung
Water
Antibodies
Brain
Enzymes
Therapeutics
Pharmaceutical Preparations

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

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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 (ECSOD), 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.",
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Cloning, purification, and characterization of recombinant human extracellular superoxide dismutase in SF9 insect cells. / Shrestha, Pravesh; Yun, Ji Hye; Kim, Woo Taek; Kim, Tae Yoon; Lee, Weon Tae.

In: Molecules and cells, Vol. 39, No. 3, 01.01.2016, p. 242-249.

Research output: Contribution to journalArticle

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