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Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: Cloning, characterization and PCR application

Paper ID Volume ID Publish Year Pages File Format Full-Text
21011 43202 2011 6 PDF Available
Title
Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: Cloning, characterization and PCR application
Abstract

The 2352 bp gene coding for 783 amino acid family B DNA polymerase from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. Expression of the gene resulted in the production of Pca-Pol in soluble fraction. After heat denaturation of the host proteins, the Pca-Pol was further purified by ion exchange and hydrophobic interaction chromatographies. Activity gel analysis showed the presence of a catalytically active polypeptide of about 90 kDa. The mass of the protein, determined by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry was found to be 89,156 Da. The isoelectric point of the enzyme was found to be 6.13. The optimal pH and magnesium ion concentration for the enzyme activity were 8.5 and 4 mM, respectively. Unlike other commercially available DNA polymerases the enzyme activity of Pca-Pol was inhibited by monovalent cations such as ammonium and potassium. The half-life of the polymerase at 95°C and 100°C was 4.5 h and 0.5 h, respectively. The enzyme possessed 3′ → 5′ exonuclease activity and was able to amplify, under suitable conditions, up to 7.5 kb DNA fragments by polymerase chain reaction which makes it a potential candidate for amplification of long DNA fragments.

Keywords
Archaea; DNA polymerase; Hyperthermophile; Pyrobaculum calidifontis; Exonuclease activity; Polymerase chain reaction
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Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: Cloning, characterization and PCR application
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 112, Issue 2, August 2011, Pages 118–123
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