title: Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production.
abstract: Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.
authors.affiliation: Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192, Japan. arimitsu@fujita‐hu.ac.jp
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Arimitsu
authors.forename: Keiko
authors.initials: K
authors.lastname: Sasaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimizu
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tsukamoto
authors.forename: Toshiyasu
authors.initials: T
authors.lastname: Shimizu
authors.forename: Takao
authors.initials: T
authors.lastname: Tsuji
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D057134:Antibodies, Neutralizing / Q000097:blood; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000633:toxicity; D001428:Bacterial Vaccines / Q000302:isolation & purification*; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008813:Mice, Inbred ICR; D010957:Plasmids; D011041:Poisoning; D022641:Shiga Toxin 2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D016019:Survival Analysis; D014121:Toxoids / Q000302:isolation & purification* / Q000633:toxicity; D014614:Vaccines, Synthetic / Q000302:isolation & purification
chemical_list: D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D022641:Shiga Toxin 2; D014121:Toxoids; D014614:Vaccines, Synthetic