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, Hyangkyu Lee, Han Choe

Research output: Contribution to journalArticle

7 Citations (Scopus)

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. 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
Volume169
Issue number5
DOIs
Publication statusPublished - 2013 Mar 1

Fingerprint

Maltose-Binding Proteins
Maltose
Disulfides
Escherichia coli
Superoxide Dismutase
Purification
Proteins
Metals
Cytoplasm
Protein Disulfide-Isomerases
Protein folding
Time and motion study
Time and Motion Studies
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Inclusion Bodies
Protein Folding
Staphylococcal Protein A
Extracellular Space
Enzyme activity
Enzymes

All Science Journal Classification (ASJC) codes

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

Cite this

Bae, Ji Young ; Koo, Bon Kyung ; Ryu, Han Bong ; Song, Jung A. ; Nguyen, Minh Tan ; Vu, Thu Trang Thi ; Son, Young Jin ; Lee, Hyangkyu ; Choe, Han. / Cu/Zn incorporation during purification of soluble human EC-SOD from E. coli stabilizes proper disulfide bond formation. In: Applied Biochemistry and Biotechnology. 2013 ; Vol. 169, No. 5. pp. 1633-1647.
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Cu/Zn incorporation during purification of soluble human EC-SOD from E. coli stabilizes proper disulfide bond formation. / Bae, Ji Young; Koo, Bon Kyung; Ryu, Han Bong; Song, Jung A.; Nguyen, Minh Tan; Vu, Thu Trang Thi; Son, Young Jin; Lee, Hyangkyu; Choe, Han.

In: Applied Biochemistry and Biotechnology, Vol. 169, No. 5, 01.03.2013, p. 1633-1647.

Research output: Contribution to journalArticle

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AU - Bae, Ji Young

AU - Koo, Bon Kyung

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AU - Song, Jung A.

AU - Nguyen, Minh Tan

AU - Vu, Thu Trang Thi

AU - Son, Young Jin

AU - Lee, Hyangkyu

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