Cloning, analysis, and expression of the gene for thermostable polyphosphate kinase of Thermus caldophilus GK24 and properties of the recombinant enzyme

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Title
Cloning, analysis, and expression of the gene for thermostable polyphosphate kinase of Thermus caldophilus GK24 and properties of the recombinant enzyme
Author(s)
H S Hoe; S K Lee; Dae Sil Lee; S T Kwon
Bibliographic Citation
Journal of Microbiology and Biotechnology, vol. 13, no. 1, pp. 139-145
Publication Year
2003
Abstract
The gene encoding Thermus caldophilus GK24 polyphosphate kinase (Tca PPK) was cloned and sequenced. The gene contains an open reading frame encoding 608 amino acids with a calculated molecular mass of 69,850 Da. The deduced amino acid sequence of Tca PPK showed a 40% homology to Escherichia coli PPK, and 39% to Klebsiella aerogenes PPK. The Tca ppk gene was expressed under the control of the T7lac promoter on pET-22b(+) in E. coli and its enzyme was purified about 70-fold with 36% yield, following heating and HiTrap chelating HP column chromatography. The native enzyme was found to have an approximate molecular mass of 580,000 Da and consisted of eight subunits. The optimum pH and temperature of the enzyme were 5.5 and 70°C, respectively. A divalent cation was required for the enzyme activity, with Mg2+ being the most effective.
Keyword
gene cloninggene expressionpolyphosphate kinaseTca PPKthermus caldophilus GK24
ISSN
1017-7825
Publisher
Korea Soc-Assoc-Inst
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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