title: [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].
abstract: Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.
authors.affiliation: Advanced Research Center for Life Science and Human, Department of Applied Molecular Chemistry, Graduate School of Industrial Technology, Nihon University, 1-2-1 Izumichou, Narashino, Chiba 275-8575, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Inoue
authors.forename: Maho
authors.initials: M
authors.lastname: Kikuchi
authors.forename: Tomoe
authors.initials: T
authors.lastname: Komoriya
authors.forename: Kunitomo
authors.initials: K
authors.lastname: Watanabe
authors.forename: Hideki
authors.initials: H
authors.lastname: Kouno
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D019976:Cloning, Organism; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins; D010738:Type C Phospholipases / Q000032:analysis / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens