Haemonchus contortus: a simple procedure for purifying surface proteins from third- and fourth-stage larvae.
Surface proteins were solubilized from exsheathed third (XL3)- and fourth (L4)-stage larvae of Haemonchus contortus by a one-step extraction procedure involving brief heat treatment of the worms in the presence of buffer and 100 mM sodium chloride. Surface proteins also could be preferentially extracted from XL3s, but not from L4s, by heating the worms briefly in 1% sodium dodecyl sulfate. The major proteins extracted by these procedures were similar in molecular weight to those detected by surface-labeling live worms with 125Iodine. Both extraction procedures solubilized a single, major protein with an apparent molecular weight of 68-97 kDa from XL3s. In contrast, extraction of L4s with 100 mM sodium chloride yielded four major proteins with relative molecular weights of 27, 29, 78, and 200 kDa. Antibodies raised in rabbits to surface proteins prepared by the sodium chloride procedure reacted with the surfaces of live worms in indirect immunofluorescence assays. The anti-XL3 surface protein serum was stage specific in immunofluorescence experiments using live worms and in immunoprecipitation experiments using 125Iodine-labeled XL3 and L4 surface proteins. The overall amino acid composition of the surface proteins is hydrophilic. Twenty-six percent of the amino acid residues of the XL3 surface proteins, which consist predominantly of the 68-97 kDa species, are glutamate or glutamine.
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Synergen, Incorporated, Boulder, Colorado 80301.
Shamansky
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Boisvenue
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006189:Haemonchus / Q000032:analysis*; D015801:Helminth Proteins / Q000032:analysis / Q000302:isolation & purification*; D007106:Immune Sera / Q000276:immunology; D007814:Larva / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D008970:Molecular Weight; D011233:Precipitin Tests; D012967:Sodium Dodecyl Sulfate; D014251:Trichostrongyloidea / Q000032:analysis*
D000596:Amino Acids; D015801:Helminth Proteins; D007106:Immune Sera; D008565:Membrane Proteins; D012967:Sodium Dodecyl Sulfate