Elsevier

Gene

Volume 53, Issue 1, 1987, Pages 97-104
Gene

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

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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.

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