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. 2013 Nov;95(11):2123-31.
doi: 10.1016/j.biochi.2013.08.005. Epub 2013 Aug 15.

Purification and characterisation of recombinant Bacteroides fragilis toxin-2

D D Kharlampieva  1 , V A Manuvera, O V Podgorny, S I Kovalchuk, O V Pobeguts, I A Altukhov, D G Alexeev, V N Lazarev, V M Govorun
Affiliations

Purification and characterisation of recombinant Bacteroides fragilis toxin-2

D D Kharlampieva et al. Biochimie. 2013 Nov.

Abstract

Fragilysin (BFT) is metalloprotease that is secreted by enterotoxigenic Bacteroides fragilis. Studying the mechanism of BFT interaction with intestinal epithelial cells requires a pure protein sample. In this study, we cloned DNA-fragments coding for the catalytic domain of fragilysin-2 and profragilysin-2 into an E. coli expression vector. Purification methods for the recombinant fragilysin-2 catalytic domain and profragilysin-2 were developed. In addition, we obtained mature active fragilysin-2 from recombinant proprotein by limited tryptic digestion. We tested the biological activity of the recombinant protein samples and revealed that E-cadherin was cleaved when HT-29 cells were treated with mature fragilysin-2 but not with profragilysin-2. Azocoll, azocasein and gelatin were not proteolytically cleaved by mature fragilysin-2. Proteins released in culture medium after HT-29 cells treatment with mature active BFT-2 were identified.

Keywords: BFT; Bacteroides fragilis; Fragilysin.

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