title: Overexpression of a hexa-histidine and T7 peptide-tagged human ribonucleotide reductase small subunit, R2 in Escherichia coli and the generation of human R2 antibodies.
abstract: Human ribonucleotide reductase (hRR) consists of two non-identical subunits: the large regulatory subunit, R1, with a molecular mass of approximately 90 kD; and the small catalytic subunit, R2, with a molecular mass of approximately 45 kD. In most human cells, R2 protein levels correlate closely with hRR holoenzyme activity. Since hRR is essential for the DNA replication and DNA repair synthesis, it has been considered as a potential target for cancer therapy. To facilitate further study of the regulation of the human R2 protein and hRR and their potential targeting for chemotherapy, a fusion protein tagged with 6 residues of histidine and an 11 amino acid sequence from the T7 capsid protein at the N-terminus of human R2 cDNA was overproduced in E. coli. The fusion protein was purified to near homogeneity and used to generate polyclonal antibodies in rabbits. The antibodies recognized the 48 kD recombinant protein and a major band of a 45 kD protein consistent with human R2 by Western blot in three human cancer cell lines including Caski, a cervix cancer cell line, KB, a nasopharyngeal cancer cell line, and KB-R, a KB derived cell line which is resistant to hydroxyurea (HU) and has a 3x overexpression of R2 protein. In contrast, the YL1/2 monoclonal antibody against yeast alpha-tubulin, which was previously reported to cross-react with human R2, required higher (>10x) concentrations and exposure times to detect the R2 protein in the 3 human cancer cell lines while also detecting two other proteins (alpha tubulin and an unidentified protein). We conclude that these new human polyclonal antibodies, with greater sensitivity and specificity than the YL 1/2 antibody, should better facilitate studies of the regulation of the human R2 protein and hRR.
authors.affiliation: UNIV WISCONSIN,SCH MED,DEPT HUMAN ONCOL,MADISON,WI 53792.
authors.forename: M
authors.initials: M
authors.lastname: Kuo
authors.forename: T
authors.initials: T
authors.lastname: Kinsella