Top DNA polymerase from Thermus thermophilus HB27 : gene cloning, sequence determination, and physicochemical properties
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- Top DNA polymerase from Thermus thermophilus HB27 : gene cloning, sequence determination, and physicochemical properties
- Joong Su Kim; Suk Hoon Koh; Jae Jong Kim; Suk Tae Kwon; Dae Sil Lee
- Bibliographic Citation
- Molecules and Cells, vol. 8, no. 2, pp. 157-161
- Publication Year
- A gene, top encoding Thermus thermophilus HB27 (Top) DNA polymerase, was cloned in E. coli and its nucleotide sequence was determined. Based on its deduced amino acid sequence, Top DNA polymerase is a 93.8 kDa protein comprising 834 amino acid residues. Top DNA polymerase showed high amino acid homology with those of other DNA polymerases from the Thermus sp., for example, 87.3% identity with Taq DNA polymerase. Codon usage in the top gene was similar to those of the proteins from other Thermus strains. The G + C content in the third position of the codons was as high as 93%. The top gene under the control of the tac promoter was expressed in E. coli [plasmid pTOP9]. DNA amplification using the recombinant Top DNA polymerase performed the same as other thermostable DNA polymerases from Thermus strains. The optimum temperature for its reaction was 76°C. An interesting observation was that the recombinant Top DNA polymerase was slowly cleaved into two fragments of about 60 kDa and 35 kDa at 4°C and -20°C. The larger fragment possessed polymerase activity like the Klenow fragment of E. coli DNA polymerase I. To prevent the cleavage of the Top DNA polymerase, a variety of protecting agents were examined. Among those examined, (NH4)2SO4 (100 mM) solution demonstrated an outstanding ability to block its cleavage for a prolonged period.
- Thermus thermophilus HB27topTop DNA Polymerase
- Korea Soc-Assoc-Inst
- Appears in Collections:
- Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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