Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody.

Publication Type:

Journal Article

Source:

Gene, Volume 53, Issue 1, p.97-104 (1987)

Keywords:

1987, Epitopes, Genetic Vectors, Humans, Hydrogen-Ion Concentration, Hypoxanthine Phosphoribosyltransferase, Immunologic Techniques, Mutation, Oligopeptides, Peptide Chain Termination, Translational, Structure-Activity Relationship, Transfection

Abstract:

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.