Expression of human DNA polymerase beta in E. coli and characterization of the recombinant protein

Further sequence analysis of the human ..beta..-polymerase cDNA revealed that the open reading frame extended 17 amino acids 5' of the start codon assigned previously. The full-length cDNA, which predicted a 335 amino acid protein, was digested with HaeIII endonuclease, and a sequence containing the entire coding region, as well as 5 nucleotides 5' of the ATG start codon, was subcloned into the expression plasmid pRC23. After induction of transformed cells the crude soluble extract contained a new protein immunoreactive with ..beta..-polymerase antibody and corresponding in size to the protein deduced from the coding region of the cDNA. About one-half of the epitope peptide in the cell was recovered in the soluble extract. This protein was easily purified to homogeneity in a yield of 1mg/50g cells. The protein was found to have about the same DNA polymerase specific activity as ..beta..-polymerase purified from mouse myeloma. The template x primer specificity and immunological properties of the cloned polymerase were similar to those of mouse ..beta..-polymerase and distinct from E. coli DNA Pol I. Substrate kinetics and other catalytic properties of the purified cloned ..beta..-polymerase were similar to those of mouse ..beta..-polymerase. These results indicate that mammalian ..beta..-polymerase is expressed intact andmore » fully active from the cloned cDNA.« less