A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic<i> Escherichia coli</i>
Submitted by Beatriz Quiñones on
PDF version| Title | A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli |
| Publication Type | Journal Article |
| Year of Publication | 2012 |
| Authors | He, X, Quiñones, B, McMahon, S, Mandrell, RE |
| Journal | Toxins Toxins (Basel)Toxins (Basel) |
| Volume | 4 |
| Pagination | 487-504 |
| Date Published | Jul |
| ISBN Number | 2072-6651 (Electronic)2072-6651 (Linking) |
| Accession Number | 22852065 |
| 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 degrees 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. |
| Short Title | ToxinsToxins |
| Alternate Journal | Toxins |
