High level of soluble expression in Escherichia coli and characterisation of the cloned Bacillus thuringiensis Cry4Ba domain III fragment.
Similar to the other known structures of Bacillus thuringiensis Cry delta-endotoxins, the crystal structure of the 65-kDa activated Cry4Ba toxin comprises three domains which are, from the N- to C-terminus, a bundle of alpha-helices, a three-beta-sheet domain, and a beta-sandwich. To investigate the properties of the C-terminal domain III in isolation from the rest of the toxin, the cloned Cry4Ba-domain III was over-expressed as a 21-kDa soluble protein in Escherichia coli, which cross-reacted with anti-Cry4Ba domain III monoclonal antibody. A highly-purified domain III was obtained in a monomeric form by ion-exchange and size-exclusion FPLC. Circular dichroism spectroscopy indicated that the isolated domain III fragment distinctly exists as a beta-sheet structure, corresponding to the domain III structure embodied in the Cry4Ba crystal structure. In vitro binding analysis via immuno-histochemical assay revealed that the Cry4Ba-domain III protein was able to bind to the apical microvilli of the susceptible Stegomyia aegypti larval midguts, albeit at lower-binding activity when compared with the full-length active toxin. These results demonstrate for the first time that the C-terminal domain III of the Cry4Ba mosquito-larvicidal protein, which can be isolated as a native folded monomer, conceivably participates in toxin-receptor recognition.
Chayaratanasin
Poramed
P
Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
Moonsom
Seangdeun
S
Sakdee
Somsri
S
Chaisri
Urai
U
Katzenmeier
Gerd
G
Angsuthanasombat
Chanan
C
D000330:Aedes; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004731:Endotoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis