The gene encoding Staphylothermus marinus DNA polymerase (Sma DNA polymerase) was cloned and sequenced. The Sma DNA polymerase gene consists of 2406 bp coding for a protein with 801 amino acid residues. The deduced amino acid sequence of Sma DNA polymerase exhibited a high degree of similarity with archaeal family B-type DNA polymerase homologues found in both crenarchaeotes and euryarchaeotes (Group I). The Sma DNA polymerase gene was expressed in Escherichia coli, 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 KCl concentrations were 14 and 80 mM, respectively. The half-life of the enzyme was determined to be 5.6 h at 95 °C and 3.6 h at 100 °C. Sma DNA polymerase possessed an associated 3′ → 5′ proofreading exonuclease activity. PCR performed with Sma DNA polymerase was found to be optimal in the presence of 30 mM Tris-HCl (pH 8.4), 2 mM MgSO4, 50 mM KCl, and 10 mM (NH4)2SO4. Under these conditions, the enzyme was capable of amplifying λ 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 Sma DNA polymerase might be useful in DNA amplification and PCR-based applications.
Bibliographical noteFunding Information:
This paper was supported by Samsung Research Fund, Sungkyunkwan University, 2004.
- DNA polymerase
- Exonuclease activity
- Polymerase chain reaction
- Staphylothermus marinus