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Produktion und Reinigung des thermolabilen Escherichia coli-Enterotoxins

Production and purification of thermolabile enterotoxin of Escherichia coli

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Zusammenfassung

Das hitzelabile Enterotoxin (LT-Toxin) ausEscherichia coli konnte in Trypticase Soy Broth mit Zusatz von 0,2% Hefe-Zellenextrakt über 12 Stunden optimal und quantitativ produziert werden. Die Behandlung der Zellen mit Polymyxin-B erhöhte die Ausbeute an LT-Toxin. Nach Anreicherung des Toxins mit Ammoniumsulfat konnte das LT-Toxin-Dialysat mittels Gelchromatographie mit AcA-34 Ultrogel von anderen Proteinen befriedigend abgetrennt werden. Als Eluent wurde 0,15 mol/l Tris-HCl-Puffer mit stufenlos steigendem pH-Wert benützt. Die Reinheit des Präparates wurde mittels Gelelektrophorese festgestellt. Die Anwesenheit des LT-Toxins in einer einzigen Fraktion wurde in vitro durch die Aktivitätsbestimmung im Rattendarm nachgewiesen (durch die Ansammlung von Flüssigkeit und Quantifizierung von cAMP in Darmflüssigkeit). Die Reproduzierbarkeit der LT-Toxin-Fraktion wurde mit einem spezifischen Radioimmunassay (RIA) und einem enzymgebundenen Immunosorbent-Analyse (ELISA) gesichert. Die LT-Fraktion hatte ein Molekulargewicht von 72 000 Dalton. Es wurden zwei Unterfraktionen des Toxins mit einem Molekulargewicht von 30 000 bzw. 40 000 Dalton gefunden.

Summary

Heat labile enterotoxin (LT toxin) ofEscherichia coli could be produced optimally and quantitatively in a trypticase soy broth with 0.2% yeast extract after 12 hours. Treatment of the cells with polymyxin B increased the yield of LT toxin. After concentration by ammonium sulphate precipitation, the LT toxin fraction was separated successfully from other proteins by Ultrogel AcA-34 gel exclusion chromatography. A 0.15 mol/l Tris-HCl buffer with gradually increasing pH was used as eluent. Purity was determined by gel electrophoresis. The presence of LT toxin in one isolated fraction was demonstrated in vitro by assaying activity in the intestines of rats (by collection of liquid and quantification of cAMP in intestinal fluid). The reproducibility of the LT fraction was ascertained by a specific radioimmunoassay (RIA) and by an enzyme-linked immunosorbent assay (ELISA). The LT fraction had a molecular weight of 72,000 dalton. Two subunits of the toxin with molecular weights of 30,000 and 40,000 dalton respectively were found.

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  1. Pharmazeutisches Institut, Abteilung für Mikrobiologie, Universität Oslo, Blindern, Postfach 11 08, Oslo 3, Norwegen

    T. Bergan & Ø. Olsvik

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  1. T. Bergan
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  2. Ø. Olsvik
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Bergan, T., Olsvik, Ø. Produktion und Reinigung des thermolabilen Escherichia coli-Enterotoxins. Infection 8 (Suppl 3), S226–S233 (1980). https://doi.org/10.1007/BF01639586

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