초고온성 고세균 써모코커스 NA1 균주로부터 새로운 고온성 DNA 중합효소

Abstract

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a family B type DNA polymerase gene (TNA1_pol) consisting of 3,927 bp. Sequence analysis revealed that the gene contained a putative 3'-5' exonuclease domain, a DNA polymerase domain and one in-frame intervening sequence of 1,605 bp (535 amino acids; intein), which is located in the middle of PolIII region. The mature form of the DNA polymerase without intein (TNA1_pol) was overexpressed in Esherichia coli, purified by metal affinity chromatography, and further characterized. Optimal activity of TNA1_pol was observed at pH 7.5 and 75 C. TNA1_pol was highly thermostable with t1/2 of 3.5 h at 100 C and 12.5 h at 95 C, respectively. The optimum condition for PCR amplification was determined, and the PCR parameters (error-rate, processivity and extension rate) of TNA1_pol were measured in comparison with rTaq, Pfu and KOD polymerases. TNA1_pol introduced 1 bp error in 4.45 kb on average in PCR amplification. The processivity of TNA1_pol was determined as 150 nt, and the extension rate of TNA1_pol was measured as 60 bases/sec, comparable to those of rTaq DNA polymerase.