Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract

Various plasmids harboring the truncated DNA polymerase gene ( polA) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable TthΔ NF2 polymerase [the gene product of polAΔ NF2, which lacked a 751-bp region (region flanked by initiation codon and FspI site in the polA gene)] was selected, and purified from the recombinant Escherichia coli. SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the TthΔ NF2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the TthΔ NF2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the TthΔ NF2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the TthΔ NF2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.