title: A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide.
abstract: The entire stx1 region from Escherichia coli O157:H7, containing two open reading frames (stx1a and stx1b), was cloned into pET-32a with a single promoter. This region was transformed into E. coli TransB (DE3), which is a trxB and gor mutation strain. After expression in the E. coli periplasm in a completely soluble form, the rStx1 was purified and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISA, and Western blot analysis. Our rStx1 have Vero cell median cytotoxic dose (CD50 ) and median lethal dose (LD50 ) values of approximately 30 ng and 1.5 µg, respectively. The final yield of the purified rStx1 ranged from 2 to 3 mg/L by one-step nickel affinity gel column chromatography. This method is an easy approach to the large-scale preparation of Stx1 at a reasonable cost.
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Tu
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Cai
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D005260:Female; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity; D017421:Sequence Analysis; D022622:Shiga Toxin 1 / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D014709:Vero Cells
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D022622:Shiga Toxin 1
keywords: native signal peptide; recombinant Shiga toxin 1(rStx1); single promoter