Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica.
A Fasciola hepatica cDNA clone of 1752 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 489 codons which encoded a 55 kDa polypeptide, showing a high degree of homology to protein disulfide isomerases. This putative antioxidant protein cDNA was expressed in Escherichia coli as a GST fusion protein. The cleaved recombinant protein was shown to be biologically active in vitro by mediating the oxidative refolding of reduced RNase. Immunoblotting studies using a specific antiserum raised against the recombinant protein showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The extracellular location of this protein was also supported by the specific immune responses found against this protein in F. hepatica experimentally infected rabbits.
Salazar-Calderón
Mara
M
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, Spain.
Martín-Alonso
José M
JM
Castro
Antonio M
AM
Parra
Francisco
F
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000032:analysis; D001483:Base Sequence; D015152:Blotting, Northern / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005210:Fasciola hepatica / Q000201:enzymology*; D005211:Fascioliasis / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D017238:Genes, Helminth; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D012260:Ribonucleases / Q000378:metabolism; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D000947:Antigens, Helminth; D018076:DNA, Complementary; D012260:Ribonucleases; D019704:Protein Disulfide-Isomerases