Production of antibodies directed against Escherichia coli helicase III and the molecular cloning of the helicase III gene.
Helicase III from Escherichia coli has been purified to near homogeneity using single-stranded DNA-dependent adenosine nucleoside 5'-triphosphatase activity as an assay to monitor the purification. The denatured form of this 18.5-kilodalton polypeptide, isolated on a preparative polyacrylamide gel run in the presence of sodium dodecyl sulfate, has been used as an antigen to direct the production of rabbit anti-helicase III antibodies. The antibodies obtained fail to inhibit directly either the helicase activity or the DNA-dependent adenosine nucleoside 5'-triphosphatase activity of helicase III. However, when the antigen-antibody complex is removed from solution by binding to Staphylococcus aureus cells with subsequent sedimentation, there is excellent correlation between the loss of both enzymatic activities and the loss of the helicase III polypeptide. The anti-helicase III antibodies have been used as a reagent to probe immunologically a library of E. coli DNA fragments inserted into the plasmid pBR322 for expression of the helicase III antigen. The gene encoding helicase III has been localized on a 2.0-kilobase pair PvuII-EcoRI fragment. Bacterial cells harboring a multicopy plasmid containing this fragment overproduce the helicase III antigen approximately 100-fold.
D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis*; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000201:enzymology*; D005260:Female; D005798:Genes, Bacterial; D011817:Rabbits
D000907:Antibodies, Bacterial; D000251:Adenosine Triphosphatases; D004265:DNA Helicases