title: A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli.
abstract: A one-step affinity chromatography method was developed to purify Shiga toxin 2 variants (Stx2) Stx2a, Stx2c, Stx2d and Stx2g from bacterial culture supernatants. Analysis of the purified Stx2 variants by denaturing gel electrophoresis revealed 32 kDa and 7 kDa protein bands, corresponding to the Stx2A- and B-subunits, respectively. However, native gel electrophoresis indicated that purified Stx2c and Stx2d were significantly higher in molecular weight than Stx2a and Stx2g. In a cytotoxicity assay with Hela cells, the 50% cytotoxic dose of Stx2a and Stx2g were 100 pg and 10 pg, respectively, but 1 ng each for Stx2c and Stx2d. Interestingly, analysis of the 50% inhibitory dose in a cell-free translational system from rabbit reticulocyte lysates indicated that Stx2g had a lower capacity to inhibit protein synthesis than the other Stx2 variants. The cytotoxicities in Hela cells were neutralized with an anti-Stx2B antibody and were denatured at 80 °C for 1 h. These findings demonstrated that Stx2 variants exhibited different toxicities, holotoxin structure, and stabilities using distinct systems for assessing toxin activities. The development of a simple method for purification of Stx2 variants will enable further studies of Stx2-mediated toxicity in various model systems.
authors.affiliation: Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Albany, CA 94710, USA. xiaohua.he@ars.usda.gov
authors.forename: Xiaohua
authors.initials: X
authors.lastname: He
authors.forename: Beatriz
authors.initials: B
authors.lastname: Quiñones
authors.forename: Stephanie
authors.initials: S
authors.lastname: McMahon
authors.forename: Robert E
authors.initials: RE
authors.lastname: Mandrell
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002474:Cell-Free System / Q000382:microbiology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D011817:Rabbits; D012156:Reticulocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D022641:Shiga Toxin 2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000633:toxicity
chemical_list: D022641:Shiga Toxin 2