RESUMO
The full thioredoxin coding sequence from Fasciola hepatica has been cloned into the pGEX-2T expression vector and produced in Escherichia coli as a fusion protein. The recombinant protein proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from F. hepatica. These observations suggest that this protein could participate in a redox cascade involved in the maintenance of cell homeostasis as well as in parasite protection against reactive oxygen species produced by the host.
Assuntos
Fasciola hepatica/metabolismo , Proteínas de Neoplasias , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Antioxidantes/farmacologia , Northern Blotting , DNA de Helmintos/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamato-Amônia Ligase/metabolismo , Immunoblotting , Insulina/metabolismo , Dados de Sequência Molecular , Oxirredução/efeitos dos fármacos , Peroxidases/metabolismo , Peroxirredoxinas , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Alinhamento de Sequência , Tiorredoxinas/genética , Tiorredoxinas/farmacologiaRESUMO
A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases. This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein. The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E. coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.
Assuntos
Antioxidantes/metabolismo , Fasciola hepatica/enzimologia , Proteínas de Neoplasias , Peroxidases/genética , Sequência de Aminoácidos , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Sequência de Bases , Northern Blotting , Southern Blotting , Western Blotting , Bovinos , Clonagem Molecular , DNA Complementar/química , DNA de Helmintos/química , Eletroforese em Gel de Poliacrilamida , Fasciola hepatica/genética , Expressão Gênica , Dados de Sequência Molecular , Fases de Leitura Aberta , Peroxidases/química , Peroxidases/metabolismo , Peroxirredoxinas , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.
