Abstract—Reactive oxygen species (ROS) have been implicated in a number of disease processes. Oxidative stress is inhibited by enzymatic defense mechanisms, such as superoxide dismutase, catalase, and glutathione peroxidase. Extracellular superoxide dismutase (EC-SOD or SOD3) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. It could potentially be used to prevent and cure cardiovascular disease, neurological disorders, and inflammation resulting from the accumulation of superoxide anions. In this study,we expressed the EC-SOD gene isolated from a human aortic smooth muscle cDNA library in food-grade Lactobacillus casei CCRC10679. We used an 18-aa signal peptide from human SOD3 (hSOD3) to mediate extracellular secretion. After electroporation and 20 rounds of erythromycin antibiotic selections, two clones were isolated that expressed high levels of recombinant EC-SOD. The recombinant protein was purified from L.casei/hSOD3 using fast protein liquid chromatography (FPLC) with a heparin affinity column. The purified enzyme showed two bands at 38- kDa (monomer) and 76-kDa(dimer) by SDS-PAGE and western blotting. The 76-kDa band was also detected by a nitrobluetetrazolium assay. Antioxidant activity of the purified EC-SOD was determined using a water-soluble tetrazolium (WST-1) assayto demonstrate that production of recombinant EC-SOD in the food-gradeL. casei actually possessed enzymatic activity.

Index Terms—Extracellular superoxide dismutase (EC-SOD), Lactobacillus casei, signal peptide, heparin, antioxidant.

Chuan-Mu Chen and Z. L. Lai are the Department of Life Sciences, National Chung Hsing University, No. 250 KuoKuang Road, Taichung 402, Taiwan (e-mail: chchen1@dragon.nchu.edu.tw, ywlai@dns.dwvs.cy.edu.tw).
Hsiao-Ling Chen is with the Department of Bioresources, Da-Yeh University, No. 168 University Road, Changhwa 515, Taiwan (e-mail: bellchen@mail.dyu.edu.tw).
C. C. Yen is with Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan (e-mail: davidcc@hotmail.com.tw).
M. L. Wang is with Department of Molecular Biotechnology, Da-Yeh University, Changhwa, Taiwan (e-mail: ling8091@yahoo.com.tw ).

 

Cite:Chuan-Mu Chen, Zi-Lun Lai, Chih-Ching Yen, Miao-Ling Wang, and Hsiao-Ling Chen, "Cloning and Expression of the Human Extracellular Superoxide Dismutase (EC-SOD) Gene in Probiotic Lactobacillus Casei," International Journal of Bioscience, Biochemistry and Bioinformatics vol. 3, no. 6, pp. 557-561, 2013.