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Cloning, expression, and partial characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii

Paper ID Volume ID Publish Year Pages File Format Full-Text
18400 42721 2006 10 PDF Available
Title
Cloning, expression, and partial characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii
Abstract

The gene encoding Sulfophobococcus zilligii DNA polymerase (Szi DNA polymerase) was cloned and sequenced. The Szi DNA polymerase gene encompasses 2394 bp, and codes for a protein consisting of 797 amino acid residues. The deduced amino acid sequence of Szi DNA polymerase exhibited a high degree of similarity with archaeal family B-type DNA polymerase homologues found in both crenarchaeotes and euryarchaeotes (Group I), and contained all of the motifs conserved in the homologues for 3′ → 5′ exonuclease and polymerase activities. The Szi DNA polymerase gene was expressed under the control of the T7lac promoter on the expression vector pET-28a(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was purified by heat treatment and Cibacron blue 3GA column chromatography. The optimum pH of the purified enzyme was determined to be in the range of 6.5–7.0. The enzyme was activated by the magnesium ion, and its activity was inhibited by EDTA and monovalent cations, such as potassium ion and ammonium ion. The half-life of the enzyme at 95 °C was calculated to be 4.5 h. Szi DNA polymerase possessed associated 3′ → 5′ and 5′ → 3′ exonuclease activities.

Keywords
DNA polymerase; Exonuclease activity; Sulfophobococcus zilligii; Archaea; Hyperthermophile
First Page Preview
Cloning, expression, and partial characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 38, Issue 6, 1 April 2006, Pages 821–830
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering