title: High-level expression of the chemically synthesized gene for microbial transglutaminase from Streptoverticillium in Escherichia coli.
abstract: We developed a novel approach for the high-level production of a microbial transglutaminase (TGase) from Streptoverticillium in E. coli. The direct expression of the TGase gene in E. coli cells did not cause overproduction, probably due to the harmful influence of TGase activity, which introduces covalent crosslinks between proteins. Therefore, we fused the chemically synthesized TGase gene coding for the entire 331 amino acid residues at the amino terminus to a bacteriophage T7 gene 10 leader peptide (260 amino acids) using an inducible expression vector. The TGase gene was expressed as inclusion bodies in the E. coli cytoplasm. Restoring 15 amino acid residues upstream of the amino terminus of the mature TGase by a two-step deletion of the fusion sequence facilitated solubilization and subsequent proteolytic cleavage, thus releasing mature TGase. Although the mature form had less TGase activity than native TGase, because of the poor refolding rate, these results suggest that this system is suitable for the efficient production of TGase.
authors.affiliation: Central Research Laboratories, Ajinomoto Co., Inc., Kawasaki, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kawai
authors.forename: S
authors.initials: S
authors.lastname: Takehana
authors.forename: H
authors.initials: H
authors.lastname: Takagi
mesh_terms: D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli; D015951:Factor Xa / Q000737:chemistry; D015870:Gene Expression; D005813:Genes, Synthetic; D002479:Inclusion Bodies; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013306:Streptomycetaceae / Q000378:metabolism; D011503:Transglutaminases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D011503:Transglutaminases; D015951:Factor Xa