Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium

Abstract

A DNA ligase gene from the hyperthermophilic bacterium Aquifex pyrophilus (Ap) was cloned and sequenced. An open reading frame of 2,157 bp that codes for a 82-kDa protein showed 40%-60% homology with a series of NAD(+)-dependent DNA ligases from different organisms. The recombinant enzyme Ap DNA ligase expressed in Escherichia coli was purified to homogeneity and characterized. The activity of Ap DNA ligase gradually increased in proportion to the concentration of monovalent salt up to 200 mM NaCl, 150 mM KCl, 200 mM NH4Cl, and 350 mM potassium glutamate. The optimum temperature and pH of Ap DNA ligase were greater than 65 degreesC and 8.0-8.6, respectively, for nick-closing activity. More than 75% of the ligation activity was retained after incubation at 95 degreesC for 60 min, whereas the half-lives of Thermus aquaticus and Escherichia coli DNA ligases at 95 degreesC were less than or equal to 15 min and 5 min, respectively. Thermostable Ap DNA ligase was applied to repeat expansion detection (RED) and could be a useful enzyme in DNA diagnostics.