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 4mM, 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 4.5h and 0.5h, 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.
authors.affiliation: School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.
authors.forename: Syed Farhat
authors.initials: SF
authors.lastname: Ali
authors.forename: Naeem
authors.initials: N
authors.lastname: Rashid
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
authors.forename: Muhammad
authors.initials: M
authors.lastname: Akhtar
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D016133:Polymerase Chain Reaction; D041543:Pyrobaculum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004259:DNA-Directed DNA Polymerase