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

Authors
 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 
Citation
 Applied Biochemistry and Biotechnology, Vol.169(5) : 1633-1647, 2013 
Journal Title
 Applied Biochemistry and Biotechnology 
ISSN
 0273-2289 
Issue Date
2013
Abstract
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. EC-SOD 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 proteindisulfide isomerase enabled the soluble overexpression of EC-SOD in the cytoplasm of E. coli. MBP-tagged human EC-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 metal incorporation and protein folding via disulfide bond.
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/86642
DOI
10.1007/s12010-012-0025-x
Appears in Collections:
1. Journal Papers (연구논문) > 3. College of Nursing (간호대학) > Dept. of Nursing (간호학과)
Yonsei Authors
이향규(Lee, Hyang Kyu)
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Full Text
http://link.springer.com/article/10.1007%2Fs12010-012-0025-x
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