Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody.
The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.
D000939:Epitopes / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007158:Immunologic Techniques; D009154:Mutation; D009842:Oligopeptides / Q000276:immunology; D010443:Peptide Chain Termination, Translational; D013329:Structure-Activity Relationship; D014162:Transfection
D000939:Epitopes; D009842:Oligopeptides; D007041:Hypoxanthine Phosphoribosyltransferase