- Expression of a Recombinant Cry1Ac Crystal Protein Fused with a Green Fluorescent Protein in Bacillus thuringiensis subsp. kurstaki Cry-B
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Jong Yul Roh , In Hee Lee , Ming Shun Li , Jin Hee Chang , Jae Young Choi , Kyung Saeng Boo , Yeon Ho Je
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J. Microbiol. 2004;42(4):340-345.
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DOI: https://doi.org/2101 [pii]
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 Abstract
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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 cry1Ac 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.
- Isolation and Characterization of a Lymantria dispar Multinucleocapsid Nucleopolyhedrovirus Isolate in Korea
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Hee Jin Shim , Jong Yul Roh , Jae Young Choi , Ming Shun Li , Soo Dong Woo , Hyun Woo Oh , Kyung Saeng Boo , Yeon Ho Je
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J. Microbiol. 2003;41(4):306-311.
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Abstract
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In Korea, a Lymantria dispar multinucleocapsid nucleopolyhedrovirus, LdMNPV-NM, was isolated and characterized from dead L. dispar larvae. The polyhedra of LdMNPV-NM were irregularly shaped with a diameter of 1.62?0.33 ?m. Numerous virions comprised of the multinucleocapsid were evident in the electron microscopic examination of the polyhedra cross sections. These polyhedra were composed of a major protein of 30 kDa. The restriction enzyme digestion patterns of LdMNPV-NM showed that this isolate had some different fragments from those of the Gypchek팜?LdMNPV isolate, although their overall profiles were similar. The deduced amino acid sequence of the enhancin gene of LdMNPV-NM showed differences when compared to previously reported enhancin genes of other LdMNPV strains. These results suggested that the LdMNPV-NM isolate from Korea was a new NPV strain and had a new enhancin gene.
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