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Toxins (Basel). 2012 Jul;4(7):487-504. doi: 10.3390/toxins4070487. Epub 2012 Jun 25.

A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli.

Author information

1
Western Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Albany, CA 94710, USA. xiaohua.he@ars.usda.gov

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.

KEYWORDS:

Shiga toxin 2 variants; cell-free translation assay; cytotoxicity; purification of Shiga toxins; thermal stability of Shiga toxins

PMID:
22852065
PMCID:
PMC3407889
DOI:
10.3390/toxins4070487
[Indexed for MEDLINE]
Free PMC Article

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