Cu/Zn Incorporation During Purification of Soluble Human EC-SOD from E. coli Stabilizes Proper Disulfide Bond Formation
| Autor: | Thu Trang Thi Vu, Han Bong Ryu, Young Jin Son, Jung A. Song, Hyangkyu Lee, Ji Young Bae, Bon Kyung Koo, Han Choe, Minh Tan Nguyen |
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| Rok vydání: | 2013 |
| Předmět: |
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Molecular Cytoplasm Protein Folding Recombinant Fusion Proteins Molecular Sequence Data Protein Disulfide-Isomerases Gene Expression Bioengineering medicine.disease_cause Applied Microbiology and Biotechnology Biochemistry Maltose-Binding Proteins Protein Structure Secondary Inclusion bodies Escherichia coli medicine Humans Amino Acid Sequence Disulfides Protein disulfide-isomerase Molecular Biology chemistry.chemical_classification biology Superoxide Dismutase Escherichia coli Proteins General Medicine Peptide Elongation Factors Enzyme assay Zinc Enzyme Solubility chemistry biology.protein Protein folding Transcriptional Elongation Factors Extracellular Space Protein A Copper Transcription Factors Biotechnology |
| Zdroj: | Applied Biochemistry and Biotechnology. 169:1633-1647 |
| ISSN: | 1559-0291 0273-2289 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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