초고온성 써모코커스 NA1의 열안정한 DNA ligase 클로닝과 특성

Abstract

An extremely thermophilic archaeal strain, NA1 was isolated from a deep-sea hydrothermal vent area at the PACMANUS field in the East Manus Basin. On the basis of 16S rRNA gene sequences, the NA1 was classified to genus, Thermococcus. As a part of genomic research of the NA1, a gene encoding DNA ligase was identified. To characterize the ligase, the gene encoding DNA ligase was cloned into Escherichia coli by PCR. The DNA ligase gene consists of 1,689 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 61,820 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved six motifs (Ⅰ, Ⅲ, Ⅲa, Ⅳ, Ⅴ, Ⅵ) suggested that NA1 DNA ligase was an ATP-dependent DNA ligase group. The deduced amino acid sequence of the NA1 DNA ligase was compared with previously reported ATP-dependent ligases, and and the result showed high similarity to other archaeal DNA ligases: T. kodakaransis KOD1 (GeneBank accession no. YP184553) (82% identity), T. fumicolans (GeneBank accession no. CAC21199) (81% identity), Pyrococcus abyssi (GeneBank accession no. CAB49465) (75% identity). The ligase was expressed Escherichia coli, and purified using histag affinity chromatography. The recombinant enzyme showed a DNA ligase activity in the presence of ATP and Mg2+.