Expression of a recombinant Cry1Ac crystal protein fused with a green fluorescent protein in Bacillus thuringiensis subsp. kurstaki Cry-B.
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.
Roh
Jong Yul
JY
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea.
Lee
In Hee
IH
Li
Ming Shun
MS
Chang
Jin Hee
JH
Choi
Jae Young
JY
Boo
Kyung Saeng
KS
Je
Yeon Ho
YH
D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism* / Q000472:pathogenicity; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization; D004731:Endotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006460:Hemolysin Proteins; D007814:Larva / Q000235:genetics / Q000378:metabolism; D007915:Lepidoptera / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D049452:Green Fluorescent Proteins