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Title 

Top DNA polymerase from Thermus thermophilus HB27 : gene cloning, sequence determination, and physicochemical properties

Authors 

Joong Su KimSuk Hoon KohJae Jong KimSuk Tae KwonDae Sil Lee

Publisher 

Springer Verlag (Germany)

Issue Date 

1998

Citation 

Molecules and Cells, vol. 8, no. 2, pp. 157-161

Keywords 

Thermus thermophilus HB27topTop DNA Polymerase

Abstract 

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.

ISSN 

1016-8478

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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