Increased Yield of High-Purity and Active Tetrameric Recombinant Human EC-SOD by Solid Phase Refolding

  • Ryu, Kang (Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea) ;
  • Kim, Young-Hoon (Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea) ;
  • Kim, Young-Hwa (Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea) ;
  • Lee, Joon-Seok (Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea) ;
  • Jeon, Byeong-Wook (Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea) ;
  • Kim, Tae-Yoon (Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea)
  • Published : 2008.10.31

Abstract

Superoxide dismutase (SOD) removes damaging reactive oxygen species from the cellular environment by catalyzing the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Extracellular superoxide dismutase (EC-SOD) is a tetramer and is present in the extracellular space and to a lesser extent in the extracellular fluids. Increasing therapeutic applications for recombinant human extracellular superoxide dismutase (rEC-SOD) has broadened interest in optimizing methods for its purification, with a native conformation of tetramer. We describe a solid phase refolding procedure that combines immobilized metal affinity chromatography (IMAC) and gel filtration chromatography in the purification of rEC-SOD from Escherichia coli. The purified rEC-SOD tetramer from the $Ni^{2+}$-column chromatography is refolded in Tris buffer. This method yields greater than 90% of the tetramer form. Greater than 99% purity is achieved with further purification over a Superose 12PC 3.2/30 column to obtain the tetramer and specific activities as determined via DCFHDA assay. The improved yield of rEC-SOD in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

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