Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis.
A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.
Klimowski
L
L
Heska Corporation, Fort Collins, CO 80525, USA.
Chandrashekar
R
R
Tripp
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004183:Dirofilaria immitis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D054464:Peroxiredoxins; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis
D018076:DNA, Complementary; D015801:Helminth Proteins; D009363:Neoplasm Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D010544:Peroxidases; D054464:Peroxiredoxins