Cloning, Nucleotide Sequencing, and Expression in Escherichia coli of DNA Polymerase Gene from The Archaea Thermococcus peptonophilus OG-1 and SM-2
Hideyuki KOMATSUBARA*a, Masao KITABAYASHIa, Bunsei KAWAKAMIa, Chiaki KATOb, and Koki HORIKOSHIc
a Tsuruga Institute of Biochemistry TOYOBO Co., LTD., 10-24 Toyo-cho, Tsuruga 914 Japan
b The DEEPSTAR group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka 237, Japan
c Department of Technology, Toyo University, 2100 Kujirai, Kawagoe-shi 350, Japan
Thermostable DNA polymerase is an indispensable enzyme for the polymerase chain reaction (PCR) method. DNA polymerase genes of many hyperthermophilic archaea have already cloned. The deduced amino acid sequences of the DNA polymerases showed that they all belong to family B. Moreover, some of these DNA polymerase genes contain two in-frame intervening protein sequence (IVPS), one of which encodes an endonuclease. In this work, we cloned the genes encoding DNA polymerases of new archaea strain, Thermococcus peptonophilus OG-1 and SM-2 (OG-1 polymerase and SM-2 polymerase, respectively) and expressed them in Escherichia coli cells. The DNA polymerase gene of OG-1 has an open reading frame of 2325 bp available to encode a peptide of 774 amino acids. The DNA polymerase gene of SM-2 has an open reading frame of 3495 bp available to encode a peptide of 1165 amino acids. SM-2 polymerase gene contains one IVPS, which was structurally homologous to the IVPS2 gene of Thermococcus litoralis encoding endonuclease. On the other hands OG-1 polymerase gene does not have IVPS. Comparison of the sequence of OG1 DNA polymerase with that of SM-2 mature DNA polymerase showed 98.3% homology at the amino acid level. These sequences showed about 80% similarity to that of mature Thermococcus litoralis (Vent polymerase). Both enzyme were extremely thermostable, having half-life of 7 hr of OG-1 polymerase and 10 hr of SM-2 polymerase at 95℃. The extension rate of OG-1 polymerase was about 60 base/sec and that of SM-2 polymerase was about 40 base/sec. The PCR fidelity of SM-2 polymerase was about 3 times higher than that of OG- 1 polymerase.
