O-019. Expression, Characterization, and Application of DNA Polymerase I Derived from Thermus kawarayensis

N. Kurosawa1, H. Matsukawa2;
1Soka Univ., Tokyo, JAPAN, 2Kyushu Univ., Fukuoka, JAPAN.

Thermus kawarayensis strain KW11 was isolated from Kawarayu hot springs, Japan. This isolate was phylogenetically related with Thermus thermophilus and Thermus aquaticus based on the 16S rDNA sequence similarity. In order to characterize DNA polymerase I of T. kawarayensis, we cloned and expressed the gene for this enzyme in E. coli. T. kawarayensis DNA polymerase I (TkaPol) was encoded by 2502 bp (833 aa, 94 kDa) as a single copy gene in the genome. The amino acid sequence of TkaPol showed identities of 89.8%, 86.9%, and 78.5% with the ones of T. thermophilus, T. aquaticus, and T. filiformis, respectively. TkaPol also had highly conserved sequence motifs which are generally found in the primary structures of bacterial DNA polymerase I. TkaPol was successfully expressed in E. coli BL21 (DE3) cells, and was purified by heat treatment, Q-sepharose and CM-sepharose column chromatography. The purified enzyme showed high specific DNA polymerase activity at 70oC, pH 7.5-8.0, and was able to be used for PCR amplification of DNA. In the optimized reaction mixture for PCR, TkaPol gave reliable amplification of DNA like commercial Taq DNA polymerase. TkaPol also exhibited reverse transcriptase activity for both synthetic RNA (poly[rA]p[dT]) and native RNA (mouse total RNA) as RNA templates. Therefore, TkaPol is useful enzyme not only for PCR but also for in vitro reverse transcription in high temperature.