| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1952167 | Biochimie | 2013 | 9 Pages |
•Recombinant profragilysin-2 and mature active fragilysin-2 (mBFT-2) were obtained.•Limited tryrptic digestion was used for conversion of profragilysin to mature form.•Loss of E-cadherin was detected after the exposure of HT-29 cells to mBFT-2.•Proteins released in culture medium after HT-29 treatment with mBFT were identified.
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.
