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Journal Article

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

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

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.

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Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.
U Fagin, U Hahn, J Grötzinger, B Fleischer, D Gerlach, F Buck, A Wollmer, H Kirchner, L Rink
Infection and Immunity Nov 1997, 65 (11) 4725-4733; DOI:

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Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.
U Fagin, U Hahn, J Grötzinger, B Fleischer, D Gerlach, F Buck, A Wollmer, H Kirchner, L Rink
Infection and Immunity Nov 1997, 65 (11) 4725-4733; DOI:
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