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KMID : 0613820080180111592
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Journal of Life Science
2008 Volume.18 No. 11 p.1592 ~ p.1599
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Cloning of the β-Lactamase Gene from Bacillus sp. J105 and Analysis of Its Expression in E. colis Cells
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Kang Won-Dae
Lim Hak-Seob
Seo Min-Jeong
Kim Min-Jeong
Lee Hye-Hyeon
Cho Kyung-Soon
Kang Byoung-Won
Seo Kwon-Il
Choi Yung-Hyun
Jeong Yong-Kee
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Abstract
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The β-lactamase gene was cloned into E. coli DH5α from Bacillus sp. J105 with strong resistance against β-lactam antibiotics. The chromosomal DNA was partially digested with Sau3AI and ligated to BamHI digested pLAFR3. β-Lactamase positive clones were obtained by using in vitro packaging kit. The pKL11-Δ4.6 with β-lactamase activity was obtained by subcloning of the recombinant plasmid (β-lac ＋). The 6.5 kb fragment in the subcloned plasmid was sequenced. The DNA fragment that contains the β-lactamase gene encodes 309 amino acids. The 0.17 kb upstream region was similar to those of B. thuringinesis and B. cereus with 97% identity. The deduced amino acids sequence was also similar to those of β-lactamase from B. thuringinesis and B. cereus with 97% and 94% identity, respectively. The phylogenetic tree also showed the relationships of the β-lactamase gene of Bacillus sp. J105 to genetically related that of other Bacillus strains. Analysis of expression pattern of the pKL11-Δ4.6 in E. coli, revealed that the secretion efficiency of β-lactamase was 4～5％ and the molecular weight was as same as that of original β-lactamase (31 kDa) from Bacillus sp. J105.
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KEYWORD
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Bacillus strains, gene cloning, β-lactam antibiotics, β-lactamase, recombinant plasmid
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