Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme.
The gene encoding Staphylothermus marinus DNA ligase (Sma DNA ligase) was cloned and sequenced. The gene contains an open reading frame consisting of 1836bp, 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 Mg(2+) and Mn(2+), and its activity was inhibited by Ca(2+) and Zn(2+). Sma DNA ligase can utilize both ATP and ADP as cofactors. The half-life of the enzyme at 100 degrees C was determined to be approximately 2.8h. The enzyme catalyzed cohesive-end intramolecular and intermolecular joining and blunt-end intermolecular joining in the presence of tricine-NaOH buffer and Mn(2+), using either ATP or ADP.
Seo
Moo Seok
MS
Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea.
Kim
Yun Jae
YJ
Choi
Jeong Jin
JJ
Lee
Mi Sun
MS
Kim
Jae Hyun
JH
Lee
Jung-Hyun
JH
Kwon
Suk-Tae
ST
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019620:Desulfurococcaceae / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011088:DNA Ligases