Open AccessReconstitution of Bordetella bronchiseptica dermonecrotic toxin from two polypeptides
Author(s) -
Kume Katsumi,
Nakai Toyotsugu
Publication year - 1987
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1987.tb02251.x
Subject(s) - dithiothreitol , bordetella bronchiseptica , antigenicity , toxin , antiserum , sephadex , trypsinization , chemistry , microbiology and biotechnology , chromatography , bordetella , biochemistry , molecular mass , biology , bacteria , bordetella pertussis , antibody , trypsin , immunology , genetics , enzyme
Dermonecrotic toxin (DNT) purified from sonic extracts of Bordetella bronchiseptica was mildly trypsinized. The trypsinized preparations were reversibly dissociated into two polypeptides, with M r s of approx. 75 000 (fragment 1) and approx. 118 000 (fragment 2), by treatment with 100 mM dithiothreitol and 6 M urea. The two fragments were separated by gel filtration on a Sephadex G‐200 column from the dissociated DNT preparations. Each of the purified fragments did not show dermonecrotizing activity for guinea pigs. The two fragments were antigenically distinct, but had partial identity with the native toxin. Whole B. bronchiseptica DNT molecule indistinguishable from the native toxin, in terms of M r , electrophoretical behavior, antigenicity, and toxicity, was reconstituted by mixing freshly purified and dialyzed fragments 1 and 2 in a molar ration of 1:1 after removal of urea and dithiothreitol by dialysis. The anti‐‘fragment 1’ antiserum completely neutralized dermonecrotizing activity of the purified DNT, whereas the anti‐‘fragment 2’ antiserum did not.