Expression of active rat DNA polymerase .beta. in Escherichia coli

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Abstract
A recombinant plasmid for expression of rat DNA polymerase .beta. was constructured in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequences for the whole rat DNA polymerase .beta.. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for .beta.-galactosidases as those for DNA polymerase .beta.. The recombinant clone, JMp.beta.5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl .beta.-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp.beta.2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp.beta.5 cells by fewer steps than the procedure for purification of DNA polymerase .beta. from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase .beta. purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp.beta.5 and the rat DNA polymerase .beta.