Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Cloning of the β-Lactamase Gene from Bacillus sp. J105 and Analysis of Its Expression in E. colis Cells

Bacillus sp. J105 유래 β-lactamase 유전자의 cloning 및 E. coli 내에서의 발현 분석

  • Kang, Won-Dae (Department of Biotechnology, Dong-A University) ;
  • Lim, Hak-Seo (Department of Biotechnology, Dong-A University) ;
  • Seo, Min-Jeong (Department of Biotechnology, Dong-A University) ;
  • Kim, Min-Jeong (Department of Biotechnology, Dong-A University) ;
  • Lee, Hye-Hyeon (Department of Biotechnology, Dong-A University) ;
  • Cho, Kyeong-Soon (Public Health and Environment Institute of Busan) ;
  • Kang, Byoung-Won (BK21 Center for Silver-Bio industrialization Project, Dong-A University) ;
  • Seo, Kwon-Il (Department of Food and Nutrition, Sunchon National University) ;
  • Choi, Yung-Hyun (Department of Biochemistry, College of Oriental Medicine, Dong-eui University) ;
  • Jeong, Yong-Kee (Department of Biotechnology, Dong-A University)
  • 강원대 (동아대학교 생명공학과) ;
  • 임학섭 (동아대학교 생명공학과) ;
  • 서민정 (동아대학교 생명공학과) ;
  • 김민정 (동아대학교 생명공학과) ;
  • 이혜현 (동아대학교 생명공학과) ;
  • 조경순 (부산광역시 보건환경연구원) ;
  • 강병원 (동아대학교 BK21 실버바이오 사업단) ;
  • 서권일 (순천대학교 식품영양학과) ;
  • 최영현 (동의대학교 한의과대학 생화학교실) ;
  • 정영기 (동아대학교 생명공학과)
  • Published : 2008.11.30
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Abstract

The $\beta$-lactamase gene was cloned into E. coli DH5$\alpha$ from Bacillus sp. J105 with strong resistance against $\beta$-lactam antibiotics. The chromosomal DNA was partially digested with Sau3AI and ligated to BamHI digested pLAFR3. $\beta$-Lactamase positive clones were obtained by using in vitro packaging kit. The pKL11-${\Delta}4.6$ with $\beta$-lactamase activity was obtained by subcloning of the recombinant plasmid ($\beta$-lac +). The 6.5 kb fragment in the subcloned plasmid was sequenced. The DNA fragment that contains the $\beta$-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 $\beta$-lactamase from B. thuringinesis and B. cereus with 97% and 94% identity, respectively. The phylogenetic tree also showed the relationships of the $\beta$-lactamase gene of Bacillus sp. J105 to genetically related that of other Bacillus strains. Analysis of expression pattern of the pKL11-${\Delta}4.6$ in E. coli, revealed that the secretion efficiency of $\beta$-lactamase was $4{\sim}5%$ and the molecular weight was as same as that of original $\beta$-lactamase (31 kDa) from Bacillus sp. J105.

$\beta$-Lactam계 항생물질에 강한 내성을 가지는 균주 Bacillus sp. J105가 생산하는 $\beta$-lactamase의 유전자를 E. coli DH5$\alpha$에 cloning하였다. Cosmid vector pLAFR3을 이용하여, Sau3AI 으로 부분 분해한 chromosomal DNA와 BamHI으로 처리한 pLAFR3을 ligation하였다. In vitro packaging kit를 사용하여 E. coli에 형질도입 하였으며 $\beta$-lactamase양성 clone주를 획득하였다. 이 recombinant plasmid ($\beta$-lac+)를 pACYC184 (4.2kb) vector를 사용하여 subcloning 하여 최종 $\beta$-lactamase의 활성이 있는 6.4 kb 단편이 포함된 pKL11${\Delta}4.6$을 제작하였다. 이 단편을 DNA 염기서열을 분석한 결과 309개의 아미노산으로 구성된 $\beta$-lactamase를 코딩하는 927 bp를 포함하고 있었다. 클로닝된 $\beta$-lactamase 유전자의 upstream을 포함하는 170 bp의 염기서열을 분석한 결과, B. thuringinesis와 B. cereus 유래의 $\beta$-lactamase 유전자의 upstream 부위와 97%의 일치를 보였다. 본 연구에서 클로닝된 $\beta$-lactamase의 아미노산을 서열을 NCBI BLAST program을 이용하여 분석해 본 결과 B. thuringinesis와 B. cereus의 $\beta$-lactamase와 각각 97%와 94%의 일치를 보였다. 또한 계통도 분석 결과 역시 본 연구에서 클로닝된 $\beta$-lactamase의 아미노산을 서열은 B. thuringinesis와 B. cereus 와 유전학적으로 아주 밀접한 관계를 보여주었다. 이 pKL11-${\Delta}4.6$를 E. coli에서 형질전환 시켜 발현 양상을 조사해 본 결과 $\beta$-lactamase의 secretion efficiency는 약 $4{\sim}5%$％였다. E. coli의 세포 내 단백질로부터 $\beta$-lactamase를 정제하여 분자량을 확인한 결과 31 kDa로 wild type의 분자량과 일치함을 확인하였다.

Keywords

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