CHARACTERIZATION OF A NEW INTRABODY DIRECTED AGAINST THE N-TERMINAL REGION OF HUMAN P53

Genes encoding the rearranged immunoglobulin heavy and light chain var iable regions of DO-1, a monoclonal antibody directed against human p5 3, have been used to construct a single-chain antibody, DO-1 recognize s 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 ex pressed in the periplasm of E. coli or at the surface of the filamento us phage M13 retained the immunological specificity and affinity of th e 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 p owerful protecting agent of p53's DNA binding activity at 37 degrees C . The DO-1 single-chain antibody has been used to construct single-cha in intracellular antibodies (intrabodies) for expression in the cytopl asm and the nucleus of mammalian cells. These anti-p53 intrabodies wer e additionally modified by addition of a C-kappa domain to increase cy toplasmic and nuclear stability, Here we show that expression of the D O-1 single-chain antibody in the H1299 cell line results in an inhibit ion 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 o f the protein.