This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fagin, U. Articles by Rink, L. Search for Related Content PubMed PubMed Citation Articles by Fagin, U. Articles by Rink, L.

 Previous Article  |  Next Article 

Infect. Immun., 11 1997, 4725-4733, Vol 65, No. 11
Copyright © 1997, American Society for Microbiology

Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli

U Fagin, U Hahn, J Grotzinger, B Fleischer, D Gerlach, F Buck, A Wollmer, H Kirchner and L Rink
Institute of Immunology and Transfusion Medicine, University of Lubeck School of Medicine, Germany.

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.

This article has been cited by other articles:

• Swietnicki, W., Barnie, A. M., Dyas, B. K., Ulrich, R. G. (2003). Zinc Binding and Dimerization of Streptococcus pyogenes Pyrogenic Exotoxin C Are Not Essential for T-cell Stimulation. J. Biol. Chem. 278: 9885-9895 [Abstract] [Full Text]  
• Okamoto, S., Kawabata, S., Nakagawa, I., Hamada, S. (2001). Administration of Superantigens Protects Mice from Lethal Listeria monocytogenes Infection by Enhancing Cytotoxic T Cells. Infect. Immun. 69: 6633-6642 [Abstract] [Full Text]