Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Amino acid sequence disclosed in whole or in part; or...
Reexamination Certificate
2000-08-11
2004-03-30
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Amino acid sequence disclosed in whole or in part; or...
C424S190100, C424S234100, C424S246100, C514S002600, C530S350000
Reexamination Certificate
active
06713063
ABSTRACT:
1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention provides new proteins for combating insects, and particularly, coleopteran, dipteran, and lepidopteran insects sensitive to the disclosed &dgr;-endotoxins derived from
Bacillus thuringiensis
. The invention provides novel chimeric crystal proteins and the chimeric cry gene segments which encode them, as well as methods for making and using these DNA segments, methods of producing the encoded proteins, methods for making synthetically-modified chimeric crystal proteins, and methods of making and using the synthetic crystal proteins.
1.2 Description of Related Art
1.2.1
B. Thuringiensis
Crystal Proteins
The Gram-positive soil bacterium
B. thuringiensis
is well known for its production of proteinaceous parasporal crystals, or &dgr;-endotoxins, that are toxic to a variety of lepidopteran, coleopteran, and dipteran larvae.
B. thuringiensis
produces crystal proteins during sporulation which are specifically toxic to certain species of insects. Many different strains of
B. thuringiensis
have been shown to produce insecticidal crystal proteins, and compositions comprising
B. thuringiensis
strains which produce proteins having insecticidal activity have been used commercially as environmentally-acceptable insecticides because of their toxicity to the specific target insect, and non-toxicity to plants and other non-targeted organisms.
Commercial formulations of naturally occurring
B. thuringiensis
isolates have long been used for the biological control of agricultural insect pests. In commercial production, the spores and crystals obtained from the fermentation process are concentrated and formulated for foliar application according to conventional agricultural practices.
1.2.2 Nomenclature of Crystal Proteins
A review by Höfte et al., (1989) describes the general state of the art with respect to the majority of insecticidal
B. thuringiensis
strains that have been identified which are active against insects of the Order Lepidoptera, i.e., caterpillar insects. This treatise also describes
B. thuringiensis
strains having insecticidal activity against insects of the Orders Diptera (i.e. flies and mosquitoes) and Coleoptera (i.e. beetles). A number of genes encoding crystal proteins have been cloned from several strains of
B. thuringiensis
. Höbfte et al. (1989) discusses the genes and proteins that were identified in
B. thuringiensis
prior to 1990, and sets forth the nomenclature and classification scheme which has traditionally been applied to
B. thuringiensis
genes and proteins. cry1 genes encode lepidopteran-toxic Cry1 proteins. cry2 genes encode Cry2 proteins that are toxic to both lepidopterans and dipterans. cry3 genes encode coleopteran-toxic Cry3 proteins, while cry4 genes encode dipteran-toxic Cry4 proteins, etc.
Recently a new nomenclature has been proposed which systematically classifies the Cry proteins based upon amino acid sequence homology rather than upon insect target specificity. This classification scheme is summarized and regularly updated in a database maintained by the
Bacillus thuringiensis
Delta-Endotoxin Nomenclature Committee at the following web site address:
epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html.
1.2.3 Mode of Crystal Protein Toxicity
All &dgr;-endotoxin crystals are toxic to insect larvae by ingestion. Solubilization of the crystal in the midgut of the insect releases the protoxin form of the &dgr;-endotoxin which, in most instances, is subsequently processed to an active toxin by midgut protease. The activated toxins recognize and bind to the brush-border of the insect midgut epithelium through receptor proteins. Several putative crystal protein receptors have been isolated from certain insect larvae (Knight et al., 1995; Gill et al., 1995; Masson et al., 1995). The binding of active toxins is followed by intercalation and aggregation of toxin molecules to form pores within the midgut epithelium. This process leads to osmotic imbalance, swelling, lysis of the cells lining the midgut epithelium, and eventual larvae mortality.
1.2.4 Molecular Biology of &dgr;-endotoxins
With the advent of molecular genetic techniques, various &dgr;-endotoxin genes have been isolated and their DNA sequences determined. These genes have been used to construct certain genetically engineered
B. thuringiensis
products that have been approved for commercial use. Recent developments have seen new &dgr;-endotoxin delivery systems developed, including plants that contain and express genetically engineered &dgr;-endotoxin genes.
The cloning and sequencing of a number of &dgr;-endotoxin genes from a variety of
Bacillus thuringiensis
strains have been described and are summarized by Höfte and Whiteley, 1989. Plasmid shuttle vectors designed for the cloning and expression of &dgr;-endotoxin genes in
E. coli
or
B. thuringiensis
are described by Gawron-Burke and Baum (1991). U.S. Pat. No. 5,441,884 discloses a site-specific recombination system for constructing recombinant
B. thuringiensis
strains containing &dgr;-endotoxin genes that are free of DNA not native to
B. thuringiensis.
The Cry1 family of crystal proteins, which are primarily active against lepidopteran pests, are the best studied class of &dgr;-endotoxins. The pro-toxin form of Cry1 &dgr;-endotoxins consist of two approximately equal sized segments. The carboxyl-half, or pro-toxin segment, is not toxic and is thought to be important for crystal formation (Arvidson et al., 1989). The amino-half of the protoxin comprises the active-toxin segment of the Cry1 molecule and may be further divided into three structural domains as determined by the recently described crystallographic structure for the active toxin segment of the Cry1Aa &dgr;-endotoxin (Grochulski et al., 1995). Domain 1 occupies the first third of the active toxin and is essential for channel formation (Thompson et al., 1995). Domain 2 and domain 3 occupy the middle and last third of the active toxin, respectively. Both domains 2 and 3 have been implicated in receptor binding and insecticidal host range activity, depending on the insect and &dgr;-endotoxin being examined (Thompson et al, 1995).
1.2.5 Chimeric Crystal Proteins
In recent years, researchers have focused effort on the construction of hybrid &dgr;-endotoxins with the hope of producing proteins with enhanced activity or improved properties. Advances in the art of molecular genetics over the past decade have facilitated a logical and orderly approach to engineering proteins with improved properties. Site-specific and random mutagenesis methods, the advent of polymerase chain reaction methodologies, and the development of recombinant methods for generating gene fusions and constructing chimeric proteins have facilitated an assortment of methods for changing amino acid sequences of proteins, fusing portions of two or more proteins together in a single recombinant protein, and altering genetic sequences that encode proteins of commercial interest.
Unfortunately, for crystal proteins, these techniques have only been exploited in limited fashion. The likelihood of arbitrarily creating a chimeric protein with enhanced properties from portions of the numerous native proteins which have been identified is remote given the complex nature of protein structure, folding, oligomerization, activation, and correct processing of the chimeric protoxin to an active moiety. Only by careful selection of specific target regions within each protein, and subsequent protein engineering can toxins be synthesized which have improved insecticidal activity.
Some success in the area, however, has been reported in the literature. For example, the construction of a few hybrid &dgr;-endotoxins is reported in the following related art:
Intl. Pat. Appl. Publ. No. WO 95/30753 discloses the construction of hybrid
B. thuringiensis
&dgr;-endotoxins for production in
Pseudomonas fluorescens
in which the non-toxic protoxin fragment of Cry1F has been replaced by the non-toxic protoxin fragment from the Cry1Ac/Cry1
Malvar Thomas
Mohan Komarlingam Sukavaneaswaran
Sivasupramaniam Sakuntala
Ball, Esq. Timothy K.
Howrey Simon Arnold & White , LLP
Monsanto Technology LLC
Navarro Mark
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