Cloning of the gene encoding DNA binding protein HU from Bacillus stearothermophilus and its expression in Escherichia coli.
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.
Kawamura
S
S
Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Kajiyama
H
H
Yamasaki
N
N
Kimura
M
M
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C040330:histone-like protein HU, bacteria