Epitope mapping of T4 endonuclease VII with monoclonal antibodies reveals importance of both ends of the protein for target binding.
Endonuclease VII (endo VII) of bacteriophage T4 is a Holliday-structure resolving enzyme that can also recognize many other defects in DNA via an altered secondary structure. The protein has a molecular mass of 18 kDa and exists as a dimer in solution. Here we report the production and characterization of monoclonal antibodies (mAbs) directed against the highly purified enzyme. From one fusion 15 hybrid cell lines producing mAbs with high affinity for endo VII could be established. The mAbs were used for epitope mapping of the protein by using N-terminal, C-terminal and internal peptides of endo VII as antigens in enzyme-linked immunoabsorbant assays. Three classes of mAbs were distinguished as follows: (1) the predominant class with 13 mAbs recognized a C-terminal epitope located between amino acid residues 115 and 145; (2) a second class, represented by one mAb, recognized an epitope located at the N terminus between amino acid residues 16 and 65; (3) a third class, represented by one mAb, recognized an epitope built from nearly the entire native protein including amino acid residues from the C and N terminus of endo VII. The latter finding suggests close proximity of the two ends, which are provided apparently by the same monomer, since the mAb from class III does also react with a mutant protein deficient in dimerization. Internal sequences of endo VII between amino acid residues 78 and 145 did not react with any of the mAbs.
Christoph
A
A
Institut für Genetik, der Universität zu Köln, Zülpicher Str. 47, Köln, D-50674, Germany.
v Heesberg
G
G
Kemper
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000145:classification / Q000276:immunology; D017122:Bacteriophage T4 / Q000201:enzymology*; D001666:Binding Sites, Antibody; D004247:DNA / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005260:Female; D006825:Hybridomas; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D011993:Recombinant Fusion Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII; D015032:Zinc