Characterization and PCR performance of a family B-type DNA polymerase from the hyperthermophilic crenarchaeon Staphylothermus marinus

Abstract

The gene encoding Staphylothermus marinus DNA polymerase (Sma DNA polymerase) was cloned and sequenced. The Sina DNA polymerase gene consists of 2406 bp coding for a protein with 801 amino acid residues. The deduced amino acid sequence of Sina DNA polymerase exhibited a high degree of similarity with archaeal family B-type DNA polymerase homologues found in both crenarchaeotes and euryarchaeotes (Group 1). The Sina DNA polymerase gene was expressed in Escherichia coh, and the expressed enzyme was then purified by heat treatment followed by three steps of chromatography. The optimum pH of the purified enzyme was 7.5, and the optimal Mg2+ and KCI concentrations were 14 and 80 mM, respectively. The half-life of the enzyme was determined to be 5.6 h at 95 degrees C and 3.6 h at 100 degrees C. Sma DNA polymerase possessed an associated 3'-> 5' proofreading exonuclease activity. PCR performed with Sina DNA polymerase was found to be optimal in the presence of 30 mM Tris-HCl (pH 8.4), 2 mM MgSO4, 50 mM KCI, and 10 mM (NH4)(2)SO4. Under these conditions, the enzyme was capable of amplifying lambda DNA fragments of up to 6 kb. The mutant frequency in PCR was 4.76% for Sma DNA polymerase, more than a 2.3-fold improvement over the 11.29% mutant frequency for Taq DNA polymerase. The results of the PCR experiments indicate that Sina DNA polymerase might be useful in DNA amplification and PCR-based applications. (C) 2006 Elsevier Inc. All rights reserved.