Cloning of the DNA polymerase gene of Bacillus caldotenax and characterization of the gene product

Abstract

The pol gene of the thermophilic eubacterium Bacillus caldotenax was cloned in a plasmid and expressed in Escherichia coli. The PCR method was used to clone the gene with no previous knowledge of the gene or protein sequence. The 3,329-bp DNA fragment containing the structural gene for DNA polymerase was sequenced. DNA polymerase, as deduced from the DNA sequence, consisted of 877 amino acids, had a molecular weight of 99,452, and was structurally homologous to the DNA polymerases of the Pol I family (family A), which includes E. coli DNA polymerase I and T7 DNA polymerase. B. caldotenax DNA polymerase (Bca polymerase) purified from the recombinant E. coli strain was characterized. Like E. coli Pol I, Bca polymerase had 5'-->3' exonuclease activity. The degraded product with the molecular weight of 65,000 was also purified and found to have polymerase activity. To overproduce this Klenow-type fragment of Bca polymerase, a recombinant expression plasmid pUI205 with a deletion in the 5'-region of the pol structural gene was constructed. The DNA polymerase produced by pUI205 is more suitable for use in the dideoxy sequencing method than the other DNA polymerases that have been used for sequencing.