Molecular Cloning and High-Level Expression of Human Polymerase β cDNA and Comparison of the Purified Recombinant Human and Rat Enzymes

Abstract The cDNA encoding the human polymerase β from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase β cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase β mRNA in Escherichia coli and all yielded a high level of human polymerase β. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters ( k cat and K m ) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.