초고온성 아케아 써모코커스 NA1으로부터 dUTPase의 특성

Abstract

As a part of genomic research of the Thermococcus sp. NA1 (TNA1), the hyperthermophilic archaea collected from a deep-sea hydrothermal vent area, a gene whose deduced amino acid sequence had high similarity to the gene encoding the dUTPase from Pyrococcus woesei and P. furiosus (83% identity and 93% similarity) was revealed. The dUTPase encoding gene comprises open reading frame of 468 bp with valine at the N-terminus, corresponding to polypeptides of 156 amino acids with predicted molecular mass of 17,679 Da and pI of 8.1. To characterize the dUTPase, the gene was cloned and overexpressed in Escherichia coli Rosetta(DE3)pLysS with the pET-24a(+) vector system. The His6-tagged TNA1 dUTPase was purified on Co2+-IMAC-Sepharose, dialyzed, and the enzyme activity was investigated. TNA1 dUTPase was capable of converting dUTP to dUMP and inorganic phosphate and enhanced the PCR in a broader range of target lengths in combination with TNA1 DNA polymerase. TNA1 dUTPase also increased the yield of products amplified with other archaeal DNA polymerases.