Immunoaffinity purification of human O6-alkylguanine-DNA alkyltransferase using newly developed monoclonal antibodies.
As well as repairing mutagenic lesions induced by simple methylating agents, O6-alkylguanine-DNA alkyltransferase repairs precursors of cytotoxic interstrand cross-links induced by chloroethylating anticancer drugs. Moreover, levels of the transferase correlate with cellular resistance to these agents. Thus far, the human transferase has not been highly purified. In our quest to obtain the homogeneous protein we have produced four stable cloned hybridomas that secrete monoclonal antibodies against the alkyltransferase from human lymphoblasts. The specificity of these monoclonals was established by immunoblot analysis and immunoprecipitation. All these antibodies recognized the alkyltransferase only after its denaturation by sodium dodecyl sulfate. One of them, designated 19.2, was used in immunoaffinity chromatography to obtain the pure protein.
Brent
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Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
von Wronski
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Pegram
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Bigner
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008780:Methyltransferases / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000911:Antibodies, Monoclonal; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase