title: Cu/Zn incorporation during purification of soluble human EC-SOD from E. coli stabilizes proper disulfide bond formation.
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 protein disulfide 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.
authors: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, 86 Asanbyeongwon-gil, Seoul 138-736, South Korea.
authors: Ji-Young
authors: JY
authors: Bae
authors: Bon-Kyung
authors: BK
authors: Koo
authors: Han-Bong
authors: HB
authors: Ryu
authors: Jung-A
authors: JA
authors: Song
authors: Minh Tan
authors: MT
authors: Nguyen
authors: Thu Trang Thi
authors: TT
authors: Vu
authors: Young-Jin
authors: YJ
authors: Son
authors: Hyang Kyu
authors: HK
authors: Lee
authors: Han
authors: H
authors: Choe
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space; D015870:Gene Expression; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; D003300:Copper; D013482:Superoxide Dismutase; D019704:Protein Disulfide-Isomerases; D015032:Zinc