ABSTRACT
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.
Subject(s)
Fasciola hepatica/metabolism , Neoplasm Proteins , Thioredoxins/metabolism , Amino Acid Sequence , Animals , Antioxidants/pharmacology , Blotting, Northern , DNA, Helminth/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Transfer Techniques , Genetic Vectors , Glutamate-Ammonia Ligase/antagonists & inhibitors , Glutamate-Ammonia Ligase/metabolism , Immunoblotting , Insulin/metabolism , Molecular Sequence Data , Oxidation-Reduction/drug effects , Peroxidases/metabolism , Peroxiredoxins , Rabbits , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Sequence Alignment , Thioredoxins/genetics , Thioredoxins/pharmacologyABSTRACT
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.
