EXCLUSION OF BIOACTIVE CONTAMINATIONS IN STREPTOCOCCUS-PYOGENES ERYTHROGENIC TOXIN-A PREPARATIONS BY RECOMBINANT EXPRESSION IN ESCHERICHIA-COLI

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesi s of a toxic shock-like syndrome and scarlet fever. Concerning its bio logical activity, mainly T-cell-stimulatory properties, conflicting da ta exist. In this study, we show that most of the SPEA preparations us ed so far contain biologically active contaminations. Natural SPEA fro m the culture supernatant of Streptococcus pyogenes NY-5 and recombina nt 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 prep arations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA clo ned in the effective high-level expression vector pIN-III-ompA2 in Esc herichia coli. The expressed SPEA shows the same amino acid compositio n as natural SPEA, whereas functional studies reported so far were car ried out,vith toxins containing an incorrect amino terminus. We descri be the rapid purification of lipopolysaccharide-, DNase-, and protease -free SPEA in two steps from the host's periplasm and its structural c haracterization by circular dichroism. Our results represent for the f irst time the production in E. coli of recombinant SPEA,vith the authe ntic N-terminal sequence and a proven superantigenic activity. Collect ively, our results indicate that immunological studies of superantigen s require highly purified substances free of biologically active conta minations.