Cu/Zn incorporation during purification of soluble human EC-SOD from E. coli stabilizes proper disulfide bond formation

Ji Young Bae, Bon Kyung Koo, Han Bong Ryu, Jung A. Song, Minh Tan Nguyen, Thu Trang Thi Vu, Young Jin Son, Hyang Kyu Lee, Han Choe

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9 Citations (Scopus)


Extracellular superoxide dismutase (EC-SOD) is the only enzyme that removes superoxide radical in the extracellular space. The reduction of EC-SOD is linked to many diseases, suggesting that the protein may have therapeutic value. ECSOD is reported to be insoluble and to make inclusion bodies when overexpressed in the cytoplasm of Escherichia coli. The refolding process has the advantage of high yield, but has the disadvantage of frequent aggregation or misfolding during purification. For the first time, this study shows that fusion with maltose-binding protein (MBP), N-utilization substance protein A, and protein disulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human ECSOD (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 hECSOD is associated with metal incorporation and protein folding via disulfide bond.

Original languageEnglish
Pages (from-to)1633-1647
Number of pages15
JournalApplied Biochemistry and Biotechnology
Issue number5
Publication statusPublished - 2013 Mar

Bibliographical note

Funding Information:
Acknowledgments This work was supported by the Korea Research Foundation (2011-0003215 and 2009-0077495) funded by the Ministry of Education, Science and Technology, the Republic of Korea and by the Asan Institute for Life Sciences (2009-307), Seoul, Korea.

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology


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