Overproduction of microbial transglutaminase in Escherichia coli, in vitrorefolding, and characterization of the refolded form

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 const ructed. Cultivation of E. coli transformed with pUCTRPMTG-02(+) or pUCTRPMT GD2 yielded a large amount of MTG (200 similar to 300 mg/liter) as insolubl e inclusion bodies. The N-terminal amino acid residue of the expressed prot ein was methionine or serine (the second amino acid residue of the mature M TG sequence), respectively. Transformed E. coli cells were disrupted, and c ollected pellets of inclusion bodies were solubilized with 8 M urea. Rapid dilution treatment of solubilized MTC restored the enzymatic activity. Refo lded MTC, 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 MTC; could be produced effici ently in E. coli.