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Cloning, expression, and partial characterization of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii by Yoon-Jin Lee; Jeong Jin Choi; Suk-Tae Kwon is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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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.

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Author

Yoon-Jin Lee; Jeong Jin Choi; Suk-Tae Kwon

Publisher

Elsevier Science; Elsevier ; Elsevier BV (ISSN 0141-0229)

Published

2006

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)