Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme

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• 作者：Moo Seok Seo ; Yun Jae Kim ; Jeong Jin Choi ; Mi Sun Lee ; Jae Hyun Kim ; Jung-Hyun Lee ; Suk-Tae Kwon
• 关键词：Staphylothermus marinus ; Sma DNA ligase ; Archaea ; Hyperthermophile
• 刊名：Journal of Biotechnology
• 出版年：2007
• 期刊代码：53_01681656
• 类别：imm
• 出版时间：20 February 2007
• 卷：128
• 期：3
• 页码：519-530
• 文件大小：648 K

摘要

The gene encoding Staphylothermus marinus DNA ligase (Sma DNA ligase) was cloned and sequenced. The gene contains an open reading frame consisting of 1836 bp, which encodes for 611 amino acid residues. Upon alignment of the entire amino acid sequence, Sma DNA ligase showed a high degree of sequence homology with the hyperthemophilic archaeal DNA ligases, 67%identity with Aeropyrum pernix K1, and 40%identity with both Pyrococcus abyssi and Thermococcus kodakarensis. An extremely high sequence identity was observed in the six conserved motifs indicative of DNA ligase. The Sma DNA ligase gene was expressed under the control of the T7lac promoter on the pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by ion exchange and metal affinity column chromatography. The enzyme was activated by both Mg²⁺ and Mn²⁺, and its activity was inhibited by Ca²⁺ and Zn²⁺. Sma DNA ligase can utilize both ATP and ADP as cofactors. The half-life of the enzyme at 100 °C was determined to be approximately 2.8 h. The enzyme catalyzed cohesive-end intramolecular and intermolecular joining and blunt-end intermolecular joining in the presence of tricine–NaOH buffer and Mn²⁺, using either ATP or ADP.