Toxins 2012, 4(7), 487-504; doi:10.3390/toxins4070487
Article

A Single-Step Purification and Molecular Characterization of Functional Shiga Toxin 2 Variants from Pathogenic Escherichia coli

Received: 14 May 2012; in revised form: 14 June 2012 / Accepted: 19 June 2012 / Published: 25 June 2012
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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: cell-free translation assay; cytotoxicity; purification of Shiga toxins; Shiga toxin 2 variants; thermal stability of Shiga toxins
PDF Full-text Download PDF Full-Text [679 KB, uploaded 25 June 2012 16:55 CEST]

Export to BibTeX |
EndNote


MDPI and ACS Style

He, X.; Quiñones, B.; McMahon, S.; Mandrell, R.E. A Single-Step Purification and Molecular Characterization of Functional Shiga Toxin 2 Variants from Pathogenic Escherichia coli. Toxins 2012, 4, 487-504.

AMA Style

He X, Quiñones B, McMahon S, Mandrell RE. A Single-Step Purification and Molecular Characterization of Functional Shiga Toxin 2 Variants from Pathogenic Escherichia coli. Toxins. 2012; 4(7):487-504.

Chicago/Turabian Style

He, Xiaohua; Quiñones, Beatriz; McMahon, Stephanie; Mandrell, Robert E. 2012. "A Single-Step Purification and Molecular Characterization of Functional Shiga Toxin 2 Variants from Pathogenic Escherichia coli." Toxins 4, no. 7: 487-504.

Toxins EISSN 2072-6651 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert