High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli
| Autor: | Daniel Bartfeld, Ziva Yavin, Kanner Dov, Tania Geller, Haim Aviv, Jacob R. Hartman, Marian Gorecki |
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| Rok vydání: | 1986 |
| Předmět: |
Genetic Vectors
Size-exclusion chromatography Ion chromatography medicine.disease_cause Superoxide dismutase Escherichia coli medicine Humans Amino Acid Sequence Cloning Molecular chemistry.chemical_classification Alanine Multidisciplinary biology Molecular mass Superoxide Dismutase Spectrum Analysis Molecular biology Recombinant Proteins Amino acid Zinc Enzyme Gene Expression Regulation chemistry Biochemistry biology.protein Apoproteins Copper Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 83:7142-7146 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichia coli by using a vector containing a thermoinducible lambda PL promoter and a beta-lactamase-derived ribosomal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented greater than 10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD. |
| Databáze: | OpenAIRE |
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