Estudos biológicos e estruturais da BthaTL, uma serinopeptidase recombinante da peçonha de Bothrops alternatus (Viperidae, Crotalinae)

• Autor(a) principal: Cardoso, Ana Flávia Vitorino
• Data de Publicação: 2005
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UFU
• Texto Completo: https://repositorio.ufu.br/handle/123456789/15695
• topic: Serinopeptidase; Bothrops alternatus; Proteína recombinante; Cobra venenosa- Veneno; Proteinase; Serina proteinases; Proteínas; CNPQ::CIENCIAS BIOLOGICAS::GENETICA

Snake venom serine proteinases usually act on the mammal hemostatic system, sometimes resembling the action of trombin, and these enzymes became objects of therapeutical interest against trombosis. In this work we describe the cloning, expression in bacterial system and partial purification of BthaTL, a recombinant serine peptidase with 233 amino acid residues from Bothrops alternatus venom. The cDNA encoding the protein was amplified by Polymerase Chain Reaction coupled with Transcriptase Reverse, reamplified by Polymerase Chain Reaction and cloned in the pGEM-T Easy vector. The cDNA was further subcloned into the pET-28a expression vector, originating pBThaTL plasmid. Transformed with the new plasmid, DH5-α and BL21(DE3) strains were been submitted to expression assays,producing the insoluble recombinant protein as inclusion bodies. Aiming its solubilization, BthaTL was denatured, and refolded in vitro for enzimatic activity recovery. The absence of activity upon fibrinogen and casein showed that the refolding protocol used was ineffective. The primary sequence deduced for the protein evidenced high degree of identity with snake venom thrombin-like serine peptidases, and it was used for the construction of a molecular model based in the three dimensional structure from Trimeresurus stejnejeri venom plasminogen activator (TSV-PA). The substitution of some essential residues in the neighborhoods of the BthaTL catalytic hole in relation to the template indicates that the two proteins do not share the same activity or specificity. Theoretical experiments of phylogenetic reconstruction had corroborated with the taxonomic classification of the snakes species, grouping the non-viperidic and viperinic enzymes in independent branches to those where the crotalinic enzymes had been located.