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Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus

Paper ID Volume ID Publish Year Pages File Format Full-Text
17187 42650 2012 6 PDF Available
Title
Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus
Abstract

We cloned and sequenced the gene encoding Thermococcus pacificus dUTPase (Tpa dUTPase). The Tpa dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. The deduced amino acid sequence of Tpa dUTPase has high sequence similarity with other archaeal dUTPases. The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. The specific activity of Tpa dUTPase on dUTP at 85 °C was 90,909 U/mg. For Tpa dUTPase activity, we determined an optimum pH of 8.5 and temperature of 85 °C. Magnesium ions strongly induced activity, with an optimum concentration of 0.75 mM. The half-life of the enzyme at 94 °C was about 7 h. The specific activity of the Tpa dUTPase on dUTP was about 10–20-fold higher than that of Tpa dUTPase on dCTP. Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.

► The Thermococcus pacificus (Tpa) dUTPase gene consists of 471 bp and encodes a 156-amino acid protein. ► The Tpa dUTPase had an 18-kDa major protein band consistent with the 17,801 Da molecular mass calculated based on the amino acid sequence. ► The specific activity of the Tpa dUTPase on dUTP was about 10–20-fold higher than that of Tpa dUTPase on dCTP. ► Tpa dUTPase enhanced the PCR amplification efficiency of long targets when Pfu and Vent DNA polymerases were used.

Keywords
Thermococcus pacificus (Tpa); dUTPase; PCR amplification
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Characterization and PCR applications of dUTPase from the hyperthermophilic euryarchaeon Thermococcus pacificus
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 51, Issues 6–7, 10 December 2012, Pages 342–347
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
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us