Genes encoding hybrid bacillus thuringiensis toxins

Details Genes encoding hybrid bacillus thuringiensis toxins Genes encoding hybrid bacillus thuringiensis toxins

2000-09-22

2004-08-24

Bugaisky, Gabriele (Department: 1653)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Genetically modified micro-organism, cell, or virus

C424S093461, C435S320100, C435S069700, C435S069100, C435S252300, C435S254110, C435S419000, C435S418000, C536S023710, C800S302000

Reexamination Certificate

active

06780408

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to hybrid toxin fragments, and toxins comprising them, derived from
Bacillus thuringiensis
insecticidal crystal proteins.
BACKGROUND OF THE INVENTION
Bacillus thuringiensis
(hereinafter B.t.) is capable of producing proteins that accumulate intra-cellularly 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 protoxins 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 midgul 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 activity 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, such 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 &agr;-helices. Domain II comprises 3 &bgr;-sheets. Domain III, the most C-terminal domain, folds into a &bgr;-sandwich. If projected on CryI sequences, domain I runs from about amino acid residues 28 to 260, domain II from about 260 to 5 460, and domain m from about 460 to 600.
DESCRIPTION OF THE INVENTION
The present invention concerns hybrid crystal proteins particularly, though not exclusively, involving CryIC together with CryIE, CryIA, or CryIG. 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 said nucleotide sequence is given in SEQ ID NO:2. The Id 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 said nucleotide sequence is given in SEQ ID NO:4. The nucleotide sequence of a B.t. CryIG gene is given in SEQ ID NO:9, and the corresponding amino acid sequence of the protein encoded by said nucleotide sequence is given in SEQ ID NO:10. These proteins are toxic to lepidopterans, but within this order of insects, each protein has different specificity. CryIC, for example, is particularly active against
S. exigua
and
M. brassicae.
According to the present invention, there is provided an isolated B.t. hybrid toxin fragment comprising at its C-terminus domain III of a first Cry protein, or a part of said domain or a protein substantially similar to said domain; and comprising at its N-terminus the N-terminal region of a second Cry protein, or a part of said region or a protein substantially similar to said region. For example, a preferred B.t. hybrid toxin fragment according to the present invention comprises at its C-terminus domain III of a first Cry protein and comprises at its N-terminus domains I and II of a second Cry protein. A preferred fragment is one that does not bind to the CryIC binding site in an insect gut when it comprises at its C-terminus domain III of CryIC, or a part of said domain or a protein substantially similar to said domain; or one that does not bind to a CryIA binding site when it comprises at its C-terminus domain m of CryIA, or a part of said domain or a protein substantially similar to said domain.
In the context of the present invention, “substantially similar” means a pure protein having an amino acid sequence that is at least 75% similar to the sequence of a protein 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 75%, 85%, 90%, or 95% similarity to each other have at least 75%, 85%, 90%, or 95% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 6 gaps, with the proviso that, with respect to the gaps, a total not more than 15 amino acid residues are affected. For the purpose of the present invention, conservative replacements may be made between amino acids within the following groups:
(i) Serine and Threonine;
(ii) Glutamic acid and Aspartic acid;
(iii) Arginine and Lysine;
(iv) Asparagine and Glutamine;
(v) Isoleucine, Leucine, Valine, and Methionine;
(vi) Phenylalanine, Tyrosine, and Tryptophan; and
(vii) Alanine and Glycine,
with the proviso that in SEQ ID NO:6, Ser and Tyr are conservative replacements at position 620, and Ala and Glu are conservative replacements at position 618; and that SEQ ID NO:8, Ser and Tyr are conservative replacements at position 627, and Ala and Glu are conservative replacements at position 625.
In the context of the present invention, “part” of a protein means a peptide comprised by said protein and having at least 80% of the consecutive sequence thereof.
In the context of the present invention, “binding site” means a site on a molecule wherein the binding between site and toxin is reversible such that the Ka between site and toxin is in the order of at least 10
4
dm
3
mole
−1
.
The toxin fragment may comprise at its N-terminus the N-terminal region of any insecticidal protein from B.t. being commonly known as “Cry” or “Cyt”, including: CryLA(a), CryIA(b) CryIA(c), CryIB, CryIC, CryID, CryIE, CryIF, CryIG, CryIH, CryIIA, CryIB, CryIIC, CryIIIA, CryIIIB, CryIIIB(b), CryIVA, CryrVB CryIVC, CryIVD, CYTA, CryX1(IIIC), CryX2(MD), CryX3, CryV, and CryX4, or a part of said region or a protein substantially similar to said region. The toxin fragment may comprise at its C-terminus domain m of CryIC, or a part of said domain or a protein substantially similar to said domain.
Thus, the fragment may comprise domain II of CryIE, CryIB, CryID, CryIA, or CryIG, or a part of said domain II or a protein substantially simidlar to said domain II, and domain m of CryIC or a part of said domain III or a protein substantially similar to said domain III. It is particularly preferred that the fragment comprises domains I and II of CryIE, CryIB, CryID, CryIA, or CryIG, or a part thereof or a protein substantially similar to said domains I and II, and domain III of CryIC or a part thereof or a protein substantially similar to said domain III.
It is most preferred that the toxin fragment comprises a region at its C-terminus comprising the sequence from amino acid position 454 to position 602 of CryIC, or a sequence substantially similar to said sequence. The fragment may comprise a region at its C-terminus comprising the sequence from amino acid position 478 to 602 of CryIC, or a sequence substantially similar to said sequence, with the proviso that if the sequence comprising amino acids 478 to 602 of CryIC is fused directly to the C-terminus of domain II of CryIA, CryIB, CryID, CryIE, or CryIG, then the folding of the fusion product is satisfactory to yield an insecticidal component of the fragment. The routineer in the art will recognize that it may be necessary to add a peptide region to the C-terminus of domain II that spaces the C-terminal region of CryIC apart, thus enabling it to fold in such a way as to exhibit insecticidal activity.
It is most p

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