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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Blotting, Western / methods
  • Cell-Free System / microbiology
  • Electrophoresis, Polyacrylamide Gel / methods
  • Escherichia coli / metabolism
  • Escherichia coli / pathogenicity*
  • HeLa Cells
  • Humans
  • Rabbits
  • Reticulocytes / cytology
  • Reticulocytes / drug effects
  • Reticulocytes / metabolism
  • Shiga Toxin 2 / genetics
  • Shiga Toxin 2 / isolation & purification*
  • Shiga Toxin 2 / metabolism*
  • Shiga Toxin 2 / toxicity


  • Shiga Toxin 2