Characterization of heat-labile uracil-DNA glycosylase from Psychrobacter sp. HJ147 and its application to the polymerase chain reaction

The gene encoding Psp HJ147 UDG (Psychrobacter sp. HJ147 uracil-DNA glycosylase) was cloned and sequenced. The gene consists of 735 bp for coding a protein with 244 amino acid residues. The deduced amino acid sequence of Psp HJ147 UDG showed a high similarity to that of Psychrobacter articus, Psychrobacter cryohalolentis K5 and Psychrobacter sp. PRwf-1. The PCR-amplified Psp HJ147 UDG gene was expressed under the control of the T7lac promoter on pTYBI in Escherichia coli BL21 (DE3). The expressed enzyme was purified with IMPACT™-CN (intein-mediated purification with an affinity chitin-binding tag) system. The optimum pH and temperature of the purified enzyme were 7.0-7.5 and 20-25 °C respectively. The optimum NaCI and KCI concentrations for the activity of the purified enzyme ranged from 50 to75 mM. The half-life of the enzyme at 50°C was approx. 45 s. These heat-labile characteristics enabled Psp HJ147 UDG to control carry-over contamination in direct PCR without loss of the PCR product. Psp HJ 147 UDG's contaminant control in both direct PCR and indirect PCR exhibited superiority over the UDG of the marine psychrophilic bacterium strain BMTU 3346 and that of E. coli.