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Cloning of the gene encoding DNA binding protein HU from Bacillus stearothermophilus and its expression in
S Kawamura1, H Kajiyama, N Yamasaki
1Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Abstract:
The gene (hbst) encoding the DNA binding protein HU from Bacillus stearothermophilus was cloned, with the Bacillus subtilis HU gene (hbsu) as a hybridization probe. The nucleotide sequence, which contains a ribosome binding site, a transcriptional termination signal, as well as the coding region, was analyzed by the dideoxy chain-termination method. The deduced amino acid sequence of an open reading frame was perfectly matched with that of B. stearothermophilus HU (BstHU) determined by the protein chemical methods [M. Kimura and K. S. Wilson, J. Biol. Chem., 258, 4007-4011 (1983)]. The gene, hbst, was overexpressed using the expression vector pET-5a in Escherichia coli, and the recombinant HU protein (r-BstHU) was purified to be homogeneity by heparin-agarose column chromatography followed by ion-exchange column chromatography on S-Sepharose. The recombinant protein thus obtained had a circular dichroism spectrum identical to that of the authentic protein and bound to DNA to the same extent as the authentic protein.
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