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Vol 45(2011) N 2 p. 229-236;
A.I. Zakabunin*, T.P. Kamynina, S.N. Khodyreva, I.A. Pyshnaya, D.V. Pyshnyi, E.A. Kchrapov, M.L. Filipenko

Gene Cloning, Purification, and Characterization of Recombinant DNA Ligases of the Thermophilic Archaea Pyrococcus abyssi and Methanobacterium thermoautotrophicum

Institute of Chemical biology and Fundamental Medicine of Siberian Branch of Russian Academy of Sciences, Novosibirsk, 630090, Russia

*zakab@ngs.ru
Received - 2010-06-25; Accepted - 2010-08-31

DNA ligase genes of the thermophilic archaeae Pyrococcus abyssi (Pab DNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned in (Escherichia coli. The resulting recombinant enzymes were tested for activity in a ligation mixture with two oligonucleotides, one containing a preformed hairpin structure. The yield of the reaction products was maximal at temperatures close to 70°C for either enzyme; their accumulation reached a plateau at 70-75% of the theoretical yield at a stoichiometric enzyme-to-substrate ratio. The enzymes differed in thermal stability. The half-life of (PabDNA ligase was approximately 60 min at 90°C. (Mth DNA ligase was completely inactivated within 10 min at this temperature. The recombinant DNA ligases from (P. abyssi and (M. thermoautotrophicum remained stabile during long-term storage at 4°С.

PabDNA ligase, MthDNA ligase, adenylation, hairpin DNA duplex, thermal stability



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