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The journal of microbiology v.42 no.4, 2004년, pp.340 - 345  
본 등재정보는 저널의 등재정보를 참고하여 보여주는 베타서비스로 정확한 논문의 등재여부는 등재기관에 확인하시기 바랍니다.

Expression of a Recombinant Cry1Ac Crystal Protein Fused with a Green Fluorescent Protein in Bacillus thuringiensis subsp. kurstaki $Cry^-B$

Roh Jong Yul    (School of Agricultural Biotechnology, Seoul National University   ); Lee In Hee    (School of Agricultural Biotechnology, Seoul National University   ); Li Ming Shun    (School of Agricultural Biotechnology, Seoul National University   ); Chang Jin Hee    (School of Agricultural Biotechnology, Seoul National University   ); Choi Jae Young    (School of Agricultural Biotechnology, Seoul National University   ); Boo Kyung Saeng    (School of Agricultural Biotechnology, Seoul National University   ); Je Yeon Ho    (School of Agricultural Biotechnology, Seoul National University  );
  • 초록

    To investigate the co-expression and crystallization of a fusion gene between the Bacillus thuringiensis crystal protein and a foreign protein in B. thuringiensis, the expression of the Cry1Ac fused with green fluorescent protein (GFP) genes in a B. thuringiensis $Cry^-B$ strain was examined. The cry1Ac gene was cloned in the B. thuringiensis-E. coli shuttle vector, pHT3101, under the control of the native cry1Ac gene promoter, while the GFP gene was inserted into the XhoI site upstream of the proteolytic cleavage site, in the middle region of the crylAc gene (pProAc-GFP). The B. thuringiensis $Cry^-B$ strain carrying pProAc-GFP (ProAc-GFP/CB) did not produce any inclusion bodies. However, the transformed strain expressed fusion protein forms although the expression level was relatively low. Furthermore, an immu­noblot analysis using GFP and Cry1Ac antibodies showed that the fusion protein was not a single spe­cies, but rather multiple forms. In addition, the N-terminal fragment of Cry1Ac and a non-fused GFP were also found in the B. thuringiensis $Cry^-B$ strain after autolysis. The sporulated cells before autolysis and the spore-crystal mixture after autolysis of ProAc-GFP/CB exhibited insecticidal activities against Plutella xylostella larvae. Accordingly, the current results suggest that a fusion crystal protein produced by the transfomant, ProAc-GFP/CB, can be functionally expressed but easily degraded in B. thuring­iensis.


  • 주제어

    Bacillus thuringiensis .   recombinant .   Cry1Ac .   GFP .   fusion protein.  

  • 참고문헌 (6)

    1. Kronstad, J.W. and H.R. Whiteley. 1986. Three classes of homologous Bacillus thuringiensis crystal protein genes. Gene 43, 29-40 
    2. Lereclus, D., H. Agaisse, M. Gominet, and J. Chaufaux. 1995. Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spoOA mutant. Bio/Technology 13, 67-71 
    3. Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward, and D.C. Prasher. 1994. Green fluorescent protein as a marker for gene expression. Science 263, 802-805 
    4. Adang, M.J., M.J. Staver, T.A. Rocheleau, J. Leighton, R.F. Barker, and D.V. Thompson. 1985. Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene 36, 289-300 
    5. Kawamura, F. and R.H. Doi. 1984. Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases. J. Bacteriol. 160, 442-444 
    6. Agaisse, H. and D. Lereclus. 1995. How does Bacillus thuringiensis produce so much insecticidal crystal protein? J. Bacteriol. 177, 6027-6032 

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