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Applied Biochemistry and Biotechnology

, Volume 162, Issue 6, pp 1585–1598 | Cite as

Expression, High Cell Density Culture and Purification of Recombinant EC-SOD in Escherichia coli

  • Young-Jin Son
  • Ji-Young Bae
  • Seon-Ha Chong
  • Hui Sun Lee
  • Sang Hyun Mo
  • Tae Yoon Kim
  • Han ChoeEmail author
Article

Abstract

Superoxide dismutase (SOD) catalyzes the dismutation of the biologically toxic superoxide anion into oxygen and hydrogen peroxide and is deployed by the immune system to kill invading microorganisms. Extracellular SOD (EC-SOD) is a copper- and zinc-containing glycoprotein found predominantly in the soluble extracellular compartment that consists of ∼30-kDa subunits. Here, we purified recombinant EC-SOD3 (rEC-SOD) from Escherichia coli BL21(DE3) expressing a pET-SOD3-1 construct. Cells were cultured by high-density fed-batch fermentation to a final OD600 of 51.8, yielding a final dry cell weight of 17.6 g/L. rEC-SOD, which was expressed as an inclusion body, comprised 48.7% of total protein. rEC-SOD was refolded by a simple dilution refolding method and purified by cation-exchange and reverse-phase chromatography. The highly purified rEC-SOD thus obtained was a mixture of monomers and dimers, both of which were active. The molecular weights of monomeric and dimeric rEC-SOD were 25,255 and 50,514 Da, respectively. The purified rEC-SOD had 4.3 EU/mg of endotoxin and the solubility of rEC-SOD was more than 80% between pH 7 and 10. In 2 L of fed-batch fermentation, 60 mg of EC-SOD (99.9% purity) could be produced and total activity was 330.24 U. The process established in this report, involving high-cell-density fermentation, simple dilution refolding, and purification with ion-exchange and reverse-phase chromatography, represents a commercially viable process for producing rEC-SOD.

Keywords

rEC-SOD Fermentation Refolding Purification MALDI-TOF 

Notes

Acknowledgment

This work was supported by Priority Research Center Program (2009-009454) and a grant (2009-0075362) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Young-Jin Son
    • 1
    • 4
  • Ji-Young Bae
    • 1
  • Seon-Ha Chong
    • 1
  • Hui Sun Lee
    • 1
  • Sang Hyun Mo
    • 2
  • Tae Yoon Kim
    • 3
  • Han Choe
    • 1
    Email author
  1. 1.Department of Physiology and Research Institute for BiomacromoleculesUniversity of Ulsan College of MedicineSeoulSouth Korea
  2. 2.Nanomol Co., LtdSeoulSouth Korea
  3. 3.Laboratory of Dermato-Immunology, College of MedicineThe Catholic University of KoreaSeoulSouth Korea
  4. 4.Biotechnology Core LaboratoryBethesdaUSA

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