Cloning, Expression and Characterization of Recombinant DNA Ligase from the Hyperthermophile, Aquifex pyrophilus
Jae-Hwan LIM*a,b, Yeon Gyu YUa, In-Geol CHOIa,b , and Ye-Sun HANa
a Structural Biology Research Center, Korea Institute of Science and technology, Seoul, Korea
b Department of Life Science, Graduate School, Korea University, Seoul, Korea
A DNA ligase from hyperthermophilic bacterium, Aquifex pyrophilus (Ap), was cloned and expressed in E. coli. Aquifex pyrophilus is a marine hyperthermophilic bacterium that grows between 67 and 95℃, with an optimum growing temperature of 85℃. It is rod-shaped with flagella and is a microaerophilic obligatory autotroph.
The recombinant enzyme, Ap DNA ligase, has been purified to homogeneity and characterized. DNA ligase is essential for the replication, repair and manipulation of DNA. There are two types of DNA ligases, those that utilize ATP as a cofactor and those that utilize NAD+. All bacterial DNA ligases are NAD+ dependent while ATP dependent enzymes are found in eukaryotes and archae. Ap DNA ligase utilizes nicotineamide adenine dinucleotide. NAD+ dependent DNA ligases have the several conserved regions, containing AMP binding site, and are very similar to size. We identified a single open reading frame of a 2157 bp, corresponding to 719 amino acids, with a molecular weight of 80 kDa. This enzyme was found to have 40-60% homology with a series of NAD+ dependent DNA ligase from different organisms. The catalytic activity of Ap DNA ligase and the relation to structure and function of this thermostable protein will be discussed.
In spite of the widespread use of DNA ligases as indispensable reagents in recombinant DNA research, very little biochemical work on their properties appeared. Thermostable DNA ligase may turn out to be technically useful tool in recombinant DNA research.
