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Precise epitope mapping of the murine transformation‐associated protein, p53.
Author(s) -
WadeEvans A.,
Jenkins J.R.
Publication year - 1985
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1985.tb03686.x
Subject(s) - biology , epitope , microbiology and biotechnology , monoclonal antibody , fusion protein , complementary dna , epitope mapping , peptide sequence , antibody , gene , recombinant dna , genetics
Murine p53 cDNA sequences were cloned into an in vitro expression vector, Protem Hind. Four deletion libraries were generated using Bal31 double‐stranded exonuclease; two being made from constructs encoding a fusion protein constructed from SV40 small t sequences and the p53 clone, p27.la; and two from the full length p53 clone, pp53‐5. Both 5′‐ and 3′‐terminal deletions of the p53 gene were made. Transcription of these constructs using Escherichia coli RNA polymerase holoenzyme, followed by translation in mRNA‐dependent rabbit reticulocyte lysate, gave in vitro, truncated protein products which were immunoprecipitated by a panel of anti‐p53 monoclonal antibodies. This approach enabled us to map accurately the binding sites of seven different monoclonal antibodies, demonstrating four distinct antigenic sites on p53. A synthetic peptide was constructed corresponding to the predicted amino acid sequence of one of these epitopes. This peptide competes with the epitope on the full length p53 protein for the relevant monoclonal antibodies and dissociates the corresponding p53/antibody complexes.