Construction and Characterization of N-Terminally Truncated DNA Polymerase from Thermus thermophilus

概要

論文の詳細を見る
Various plasmids harboring the truncated DNA polymerase gene (polA) from Thermus thermophilus HB8 (Tth polymerase) were constructed. The most thermostable Tth△NF2 polymerase [the gene product of polA △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 Tth△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 Tth△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℃. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth△NF2 polymerase at 70℃ for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth△NF2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3'-hydroxyl group of blunt-ended DNA.
社団法人日本生物工学会の論文
1996-06-25