Molecular Cloning of the Gene for Microbial Transglutaminase fromStreptoverticilliumand Its Expression inStreptomyces lividans

Bacterial pro‐transglutaminase from Streptoverticillium mobaraense

“…mobaraense detected by using antibodies raised against the mature enzyme. The structure of the pro-region was determined by protein and nucleotide sequencing which modified data published by Washizu et al [11]. The physiological role of bacterial transglutaminase is discussed on the basis of our studies with different cell fractions.…”

“…Sequence analysis by Edman degradation of strain S-8112, which has been identified as a variant of Sv. mobaraense [11], revealed a protein consisting of 331 amino acids with a molecular mass of 37.9 kDa [12]. In contrast to other transglutaminases, only a single cysteine was determined, located at the active site.…”

“…The primary structure derived from genomic DNA confirmed the sequence of the mature protein which is quite different from all other published transglutaminases. Additionally, analysis of the entire coding region suggests that bacterial transglutaminase was synthesised as prepro-enzyme consisting of 406 amino acid residues [11]. According to the authors, the prepro-region is composed of a hydrophobic strain of 18 amino acids at the amino terminal, possibly the signal peptide, and a polypeptide of 57 amino acids for the proregion which was postulated to be important for efficient protein secretion or folding.…”

High-level Expression of the Chemically Synthesized Gene for Microbial Transglutaminase from Streptoverticillium in Escherichia coli

“…7) The previous studies showed that, based on the primary structure of the TGase, the gene encoding TGase has been cloned from the genome of Strep toverticillium S-8112 using a polymerase chain reaction amplified fragment as a probe, and it was expressed using the Streptomyces lividans host-vector system under a tyrosinase promoter. 8) In addition, the gene encompassing the entire coding region for the protein was chemically synthesized, and the gene product was secreted into the periplasmic space of E. coli using an expression vector, pIN-III-ompA, which carries an ompA signal peptide, and was identical with the native TGase in size and immunological properties. 9 ) This TGase is of interest not only in regard to its structure-function relationships, but also to its applications for food processing 10 14) and medical use.…”

“…9 ) On the other hand, productivity of TGase from the cloned gene in Streptomyces lividans carrying the expression plasmid was only 0.1 mg/liter, much lower than the productivity from the original producing strain. 8 ) In this study, we describe the construction of a high-level expression system of the chemically synthesized TGase gene in E. coli. For this purpose, it was effective to produce an inactive form as a fusion protein with a bacteriophage T7 gene 10 leader peptide.…”