Top DNA polymerase from Thermus thermophilus HB27: gene cloning, sequence determination, and physicochemical properties.
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 degrees 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 degrees C and -20 degrees 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.
Kim
J S
JS
Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST, Taejon, Korea.
Koh
S
S
Kim
J J
JJ
Kwon
S T
ST
Lee
D S
DS
D000595:Amino Acid Sequence; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D004795:Enzyme Stability; D015870:Gene Expression / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*
D004259:DNA-Directed DNA Polymerase