The EC-SOD cDNA was cloned by polymerase chain reaction (PCR) and inserted into theEscherichia coli expression plasmid pET-28a(+) and transformed into E.coli BL21 (DE3).The correspondingprotein that was overexpressed as a recombinant His_6-tagged EC-SOD was present in the form of inactiveinclusion bodies.This structure was first solubilized under denaturant conditions (8.0 M urea).Then,after acapture step using immobilized metal affinity chromatography (IMAC),a gradual refolding of the protein wasperformed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione(GSH)and oxidized glutathione(GSSG).The mass ratio of GSH to GSSG was 4:1.The purified enzyme was active,showing that at least part of the protein was properly refolded.The protein was made concentrated byultrafiltration,and then isolated using Sephacryl S-200 HR.There were two protein peaks in the A_(280) profile.Based on the results of electrophoresis,we concluded that the two fractions were formed by protein subunitsof the same mass,and in the fraction where the molecular weight was higher,the dimer was formed throughthe disulfide bond between subunits.Activities were detected in the two fractions,but the activity of thedimer was much higher than that of the single monomer.The special activities of the two fractions werefound to be 3475 U/mg protein and 510 U/mg protein,respectively. The corresponding protein was cloned by polymerase chain reaction (PCR) and inserted into the Escherichia coli expression plasmid pET-28a (+) and transformed into E. coli BL21 (DE3). The corresponding protein that was overexpressed as a recombinant His_6- tagged EC -SOD was present in the form of inactiveinclusion bodies.This structure was first solubilized under denaturant conditions (8.0 M urea). Then, after acapture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein wasperformed on-column using a linear urea gradient from 8.0 M to 1.5 M in the presence of glutathione (GSH) and oxidized glutathione (GSSG). The mass ratio of GSH to GSSG was 4: 1. The purified enzyme was active, showing that at least part of the protein was properly refolded. The protein was made concentrated by ultra filtration, and then isolated using Sephacryl S-200 HR. There were two protein peaks in the A_ (280) profile. Based on the results of electrophoresis, we concluded that the two fractions were fo rmed by protein subunits of the same mass, and in the fraction where the molecular weight was higher, the dimer was formed through the disulfide bond between subunits. Activities were detected in the two fractions, but the activity of theimer was much higher than that of the single The special activities of the two fractions were fined to be 3475 U / mg protein and 510 U / mg protein, respectively.