Cu/Zn Incorporation During Purification of Soluble Human EC-SOD from E. coli Stabilizes Proper Disulfide Bond Formation

Abstract

Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction ofEC-SODis linked to many diseases, suggesting that the protein may have therapeutic value.EC-SODis reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichiacoli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding duringpurification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and proteindisulfideisomerase enabled thesolubleoverexpression ofEC-SODin the cytoplasm ofE.coli. MBP-taggedhumanEC-SOD(hEC-SOD) was purified by MBP affinity and anion exchange chromatography, and its identity was confirmed by MALDI-TOF MS analysis. The purified protein showed good enzyme activity in vitro; however, there was a difference in metal binding. When copper and zinc were incorporated into hEC-SOD before MBP tag cleavage, the enzymatic activity was higher than when the metal ions were bound to the purified protein after MBP tag cleavage. Therefore, the enzymatic activity of hEC-SOD is associated with metalincorporationand protein folding viadisulfidebond.