title: Purification and characterisation of recombinant Bacteroides fragilis toxin-2.
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.
authors: Research Institute for Physico-Chemical Medicine of the Federal Medical and Biological Agency of Russian Federation, Malaya Pirogovskaya Str. 1a, Moscow 119435, Russia. Electronic address: harlampieva_d@mail.ru.
authors: D D
authors: DD
authors: Kharlampieva
authors: V A
authors: VA
authors: Manuvera
authors: O V
authors: OV
authors: Podgorny
authors: S I
authors: SI
authors: Kovalchuk
authors: O V
authors: OV
authors: Pobeguts
authors: I A
authors: IA
authors: Altukhov
authors: D G
authors: DG
authors: Alexeev
authors: V N
authors: VN
authors: Lazarev
authors: V M
authors: VM
authors: Govorun
mesh_terms: D001391:Azo Compounds / Q000737:chemistry; D001441:Bacteroides fragilis / Q000737:chemistry / Q000235:genetics*; D015820:Cadherins / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D003094:Collagen / Q000737:chemistry; D004926:Escherichia coli; D005780:Gelatin / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D019073:HT29 Cells; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001391:Azo Compounds; D015820:Cadherins; D002364:Caseins; C026018:azocasein; C028470:Azocoll; D005780:Gelatin; D003094:Collagen; D008666:Metalloendopeptidases; C091198:fragilysin
keywords: BFT; Bacteroides fragilis; Fragilysin