Production of polyclonal antisera to choline acetyltransferase using a fusion protein produced by a cDNA clone.
A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001329:Autolysis; D002795:Choline O-Acetyltransferase / Q000235:genetics / Q000276:immunology*; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007106:Immune Sera / Q000276:immunology*; D007118:Immunoassay; D007163:Immunosorbent Techniques; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*; D001616:beta-Galactosidase
D000941:Antigens; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004247:DNA; D002795:Choline O-Acetyltransferase; D001616:beta-Galactosidase