Overproduction of Microbial Transglutaminase in Escherichia coli, In Vitro Refolding, and Characterization of the Refolded Form

概要

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The Streptoverticillium transglutaminase (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.coli transformed with pUCTRPMTG-02(+) or pUCTRPMTGD2 yielded a large amount of MTG (200〜300mg/liter) as isoluble 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.coli cells were disrupted, and collected pellets of inclusion bodies were solubilized with 8M 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.
社団法人日本農芸化学会の論文
2000-06-23