Characterization of a new intrabody directed against the N-terminal region of human p53.
Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of DO-1, a monoclonal antibody directed against human p53, have been used to construct a single-chain antibody. DO-1 recognizes an N-terminal epitope in the region involved in the transactivation function of p53 and the binding of Mdm2. The DO-1 single chain scFv expressed in the periplasm of E. coli or at the surface of the filamentous phage M13 retained the immunological specificity and affinity of the full length antibody. Furthermore, the DO-1 recombinant antibody was able to inhibit the in vitro binding of Hdm2, and was shown to be a powerful protecting agent of p53's DNA binding activity at 37 degrees C. The DO-1 single-chain antibody has been used to construct single-chain intracellular antibodies (intrabodies) for expression in the cytoplasm and the nucleus of mammalian cells. These anti-p53 intrabodies were additionally modified by addition of a Ckappa domain to increase cytoplasmic and nuclear stability. Here we show that expression of the DO-1 single-chain antibody in the H1299 cell line results in an inhibition of p53's transactivation function. The DO-1 intrabody is a useful tool to study those functions of p53 driven by the N-terminal region of the protein.
Cohen
P A
PA
Department of Biochemistry, Medical Sciences Institute, University of Dundee, UK.
Mani
J C
JC
Lane
D P
DP
D000906:Antibodies; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D001943:Breast Neoplasms / Q000473:pathology; D002467:Cell Nucleus / Q000276:immunology / Q000378:metabolism; D003593:Cytoplasm / Q000276:immunology / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D000939:Epitopes / Q000276:immunology*; D005260:Female; D005803:Genes, Immunoglobulin; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008175:Lung Neoplasms / Q000473:pathology; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000276:immunology*; D015202:Protein Engineering; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000276:immunology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; C055966:immunoglobulin Fv; D004247:DNA; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2