SELECTIVE DNA-BINDING OF THE HUMAN CELLULAR MYB-PROTEIN ISOLATED BY IMMUNOAFFINITY CHROMATOGRAPHY USING A MONOCLONAL-ANTIBODY

  • 1 January 1987
    • journal article
    • research article
    • Vol. 1  (4) , 395-401
Abstract
The bacterially expressed v-myb protein served as antigen for the isolation of several monoclonal antibodies, one of which recognized the human cellular myb protein (p75hu-c-myb) indicating a conserved epitope. The epitope was mapped to amino acid positions 208-232 by the use of several bacterially expressed v-myb proteins with various deletions. Furthermore, a synthetic oligopeptide which had been selected on the basis of its hydrophilicity by computer analysis of the v-myb oncogene (amino acids 213-231) blocked the action of this monoclonal antibody, indicating the immunological significance of this region. The monoclonal antibody allowed efficient purification of the p75hu-cmyb protein by immunoaffinity chromatography. The purified protein binds to double-stranded DNA in vitro in a filter-binding assay. Since the monoclonal antibody does not interfere with DNA binding it allowed analysis of DNA-protein interaction in a modified McKay assay using the purified p75hu-c-myb protein. Specific binding was observed predominantly to one of 12 .lambda. DNA fragments in vitro in the presence of high molar excess of competing co-polymer poly [d(I:C)]. Enhancer/promoter-like sequences of SV40 were not preferentially recognized.