| Autor: |
YOKOYAMA, Kei-ichi, NAKAMURA, Nami, SEGURO, Katsuya, KUBOTA, Kouji |
| Zdroj: |
Bioscience, Biotechnology, and Biochemistry; January 2000, Vol. 64 Issue: 6 p1263-1270, 8p |
| Abstrakt: |
The Streptoverticilliumtransglutaminase (MTG) gene, synthesized previously for yeast expression, was modified and resynthesized for overexpession in E. coli. A high-level expression plasmid, pUCTRPMTG-02(+), was constructed. Furthermore, to eliminate the N-terminal methionine, pUCTRPMTGD2 was constructed. Cultivation of E. colitransformed with pUCTRPMTG-02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200~300 mg/liter) as insoluble inclusion bodies. The N-terminal amino acid residue of the expressed protein was methionine or serine (the second amino acid residue of the mature MTG sequence), respectively. Transformed E. colicells were disrupted, and collected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTG restored the enzymatic activity. Refolded MTG, purified by ion-exchange chromatography, which had an N-terminal methionine or serine residue, showed activity equivalent to that of native MTG. These results indicated that recombinant MTG could be produced efficiently in E. coli. |
| Databáze: |
Supplemental Index |
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