Overproduction of microbial transglutaminase in Escherichia coli, in vitro refolding, and characterization of the refolded form.
(MTG) The Streptoverticillium transglutaminase gene, synthesized previously for yeast expression, was modified and resynthesized for overexpression 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 pUCTRPMTG02(+) 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. coli cells 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.
Yokoyama
K I
KI
Central Research Laboratories, Ajinomoto Co. Inc, Kawasaki, Kanagawa, Japan. Keiichia_yokoyama@ajinomoto.com
Nakamura
N
N
Seguro
K
K
Kubota
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013306:Streptomycetaceae / Q000201:enzymology* / Q000235:genetics; D011503:Transglutaminases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D011503:Transglutaminases