Cloning, Expression, and Characterization of a DNA Ligase from a Hyperthermophilic Archaeon Thermococcus Sp.

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. NA1, revealed an ORF of 1689 bases encoding 562 amino acids that showed a high similarity to DNA ligases from other hyperthermophilic archaea. The ligase, which was designated TNA1_lig ( Thermococcus sp. NA1 ligase), was cloned and expressed in Escherichia coli. The recombinant TNA1_lig was purified by metal affinity chromatography. The optimum ligase activity of the recombinant TNA1_lig occurred at 80 °C and pH 7.5. The enzyme was activated by MgCl₂ and ZnCl₂ but was inhibited by MnCl₂ and NiCl₂. Additionally, the enzyme was activated by either ATP or NAD⁺. [ABSTRACT FROM AUTHOR]