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

Paper ID Volume ID Publish Year Pages File Format Full-Text
25272 43564 2007 12 PDF Available
Title
Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme
Abstract

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.

Keywords
Staphylothermus marinus; Sma DNA ligase; Archaea; Hyperthermophile
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Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 128, Issue 3, 20 February 2007, Pages 519–530
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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Full-text PDF Download
Online Support
Any Questions? feel free to contact us