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

다국어 초록 (Multilingual Abstract)

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.

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