Hybrid toxin

Details Hybrid toxin Hybrid toxin

1996-02-21

1998-04-07

Wax, Robert A.

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

424 94461, 424 931, 514 12, 514 2, 435 697, 4351723, 4353201, 4352523, 43525231, 43525411, 800200, 800205, 800230, 800250, 530350, 536 2371, 536 234, A01N 6300, A01N 3718, C07K 14325, C12P 2102

Patent

active

057361312

DESCRIPTION:

BRIEF SUMMARY
The present invention relates to hybrid toxin figments, and toxins comprising them, derived from Bacillus thuringiensis insecticidal crystal proteins.
Bacillus thuringiensis (hereinafter B.t.) is capable of producing proteins that accumulate intracellularly as crystals. These crystal proteins are toxic to a number of insect larvae. Based on sequence homology and insecticidal specificity, crystal proteins have been categorized into different classes. Best studied are the CryI class of proteins which are produced as 140 kDa pro-toxins and are active towards lepidopterans.
To some extent the mode of action of crystal proteins has been elucidated. After oral uptake the crystals dissolve in the alkaline environment of the larval midgut. The solubilized proteins are subsequently processed by midgut proteinases to a proteinase-resistant toxic fragment of about 65 kDa which binds to receptors on epithelial cells of the insect midgut and penetrates the cell membrane. This eventually leads to bursting of the cells and death of the larvae.
The activity spectrum of a particular crystal protein is to a large extent determined by the occurrence of receptors on the midgut epithelial cells of susceptible insects. The said spectrum is co-determined by the efficiency of solubilization of the crystal protein and its proteolytic activation in vivo.
The importance of the binding of the crystal protein to midgut epithelial receptors is further demonstrated where insects have developed resistance to one of the crystal proteins in that the binding of crystal proteins to midgut epithelial cells in resistant insects is significantly reduced.
Toxic fragments of crystal proteins are thought to be composed of three distinct structural domains. Domain I, the most N-terminal domain, consists of 7 .alpha.-helices. Domain II comprises 3 .beta.-sheets and domain III (the most C-terminal) folds into a .beta.-sandwich. If projected on CryI sequences, domain I runs from about amino acid residue 28 to 260; domain II from about 260 to 460 and domain III from about 460 to 600.
The present invention concerns hybrid crystal proteins particularly, though not exclusively, involving CryIC and CryIE or CryIA. The nucleotide sequence of the CryIC gene from B.t. sub.sp. entomocidus 60.5 is given in SEQ ID. No. 1, and the corresponding amino acid sequence of the protein encoded by the said nucleotide sequence is given in SEQ ID No. 2. The nucleotide sequence of the CryIE gene from B.t. sub. sp. kenyae 4FI is given in SEQ ID. No. 3, and the corresponding amino acid sequence of the protein encoded by the said nucleotide sequence is given in SEQ ID No. 4. These proteins are toxic to lepidopterans, but within this order of insects, each protein has different specificity. CryIC is particularly active against S. exigua and M. brassicae.
According to the present invention there is provided a B.t. hybrid toxin fragment comprising at its C-terminal domain III of a first Cry protein or a part of the domain or a protein substantially similar to the domain, with the proviso that the N-terminal region of the fragment is the N-terminal region of a second Cry protein or a part of the region or a protein substantially similar to the region. A preferred fragment is one which does not bind to the CryIC binding site in an insect gut when it comprises at its C-terminal domain III of CryIC or a part of the domain or a protein substantially similar to the domain; or one which does not bind to a CryIA binding site when it comprises at its C-terminal domain III of CryIA or a part of the domain or a protein substantially similar to the domain.
By substantially similar is meant pure proteins having an amino acid sequence which is at least 75% similar to the sequence of the proteins according to the invention. It is preferred that the degree of similarity is at least 85%, more preferred that the degree of similarity is at least 90% and still more preferred that the degree of similarity is at least 95%.
In the context of the present invention, two amino acid sequences with at least

REFERENCES:
Honee et al. The C-terminal domain of the toxic fragment of a Bacillus thuringiensiscrystal protein determines receptor binding. Molecular Microbiology 5(11)2799-2806, 1991.
Nakamura et al. Construction of chimeric insecticidal crystal proteins between the 130 kda amd 135 kda proteins of Bacillus thuringiensis subsp. aizawai for analysis of structure-function relationship. Agric. Biol. Chem. 54(3): 715-724 Mar. 1990.
Ge et al. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis .delta.o-endotoxin protein. Proc. Nat. Acad. Sci., USA 86: 4037-4041, Jun. 1989.
Bosch et al. Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Bio/technology 12: 915-918, Sep. 12, 1994.
Visser et al. Domian--function studies of Bacillus thuringiensis crystal proteins: a genetic approach. in Bacillus thuringiensis, an environmental biopesticide: theory and practice. (eds, Entwistle et al.) Chicester: Wiley & Sons 1993.

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