Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2001-01-05
2003-10-28
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S417000, C435S418000, C424S171100, C436S513000, C504S116100
Reexamination Certificate
active
06639054
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to non-naturally occurring novel proteins which have insecticidal properties, and more specifically to the design, preparation, and use of proteins that have been deallergenized while maintaining their insecticidal properties. Deallergenized patatin proteins include variants that have had allergenic sequences modified, and permuteins that have had their amino acid sequences rearranged at one or more breakpoints.
BACKGROUND OF THE INVENTION
Insecticidal Proteins
The use of natural products, including proteins, is a well known method of controlling many insect, fungal, viral, bacterial, and nematode pathogens. For example, endotoxins of
Bacillus thuringiensis
(
B.t
.) are used to control both lepidopteran and coleopteran insect pests. Genes producing these endotoxins have been introduced into and expressed by various plants, including cotton, tobacco, and tomato. There are, however, several economically important insect pests such as boll weevil (BWV),
Anthonomus grandis
, and corn rootworm (CRW), Diabrotica spp. that are not as susceptible to
B.t
. endotoxins as are insects such as lepidopterans. In addition, having other, different gene products for control of insects which are susceptible to
B.t
. endotoxins is important, if not vital, for resistance management.
It has been recently discovered that the major storage protein of potato tubers, patatins (Gaillaird, T.,
Biochem. J
. 121: 379-390, 1971; Racusen, D.,
Can. J. Bot
., 62: 1640-1644, 1984; Andrews, D. L., et al.,
Biochem. J
., 252: 199-206, 1988), will control various insects, including western rootworm (WCRW,
Diabrotica virigifera
), southern corn rootworm (SCRW,
Diabrotica undecimpunctata
), and boll weevil (BWV,
Anthonomus grandis
) (U.S. Pat. No. 5,743,477). Patatins are lethal to some larvae and will stunt the growth of survivors so that maturation is prevented or severely delayed, resulting in no reproduction. These proteins, have nonspecific lipid acyl hydrolase activity and studies have shown that the enzyme activity is essential for its insecticidal activity (Strickland, J. A., et al.,
Plant Physiol
., 109: 667-674, 1995; U.S. Pat. No. 5,743,477). Patatins can be applied directly to the plants or introduced in other ways well known in the art, such as through the application of plant-colonizing microorganisms, which have been transformed to produce the enzymes, or by the plants themselves after similar transformation.
In potato, the patatins are found predominantly in tubers, but also at much lower levels in other plant organs (Hofgen, R. and Willmitzer, L.,
Plant Science
, 66: 221-230, 1990). Genes that encode patatins have been previously isolated by Mignery, G. A., et al. (
Nucleic Acids Research
, 12: 7987-8000, 1984; Mignery, G. A., et al.,
Gene
, 62: 27-44, 1988; Stiekema, et al.,
Plant Mol. Biol
., 11: 255-269, 1988) and others. Patatins are found in other plants, particularly solanaceous species (Ganal, et al.,
Mol. Gen. Genetics
, 225: 501-509, 1991; Vancanneyt, et al.,
Plant Cell
, 1: 533-540, 1989) and recently
Zea mays
(WO 96/37615). Rosahl, et al. (
EMBO J
., 6: 1155-1159, 1987) transferred it to tobacco plants, and observed expression of patatin, demonstrating that the patatin genes can be heterologously expressed by plants.
Patatin is an attractive for use in planta as an insect control agent, but unfortunately a small segment of the population displays allergic reactions to patatin proteins, and in particular to potato patatin, as described below.
Food Allergens
There are a variety of proteins that cause allergic reactions. Proteins that have been identified as causing an allergic reaction in hypersensitive patients occur in many plant and animal derived foods, pollens, fungal spores, insect venoms, insect feces, and animal dander and urine (King, H. C.,
Ear Nose Throat J
., 73(4): 237-241, 1994; Astwood, J. D., et al.,
Clin. Exp. Allergy
, 25: 66-72, 1995; Astwood, J. D. and Fuchs R. L.,
Monographs in allergy Vol
. 32
: Highlights in food allergy
, pp. 105-120, 1996; Metcalfe, D. D., et al.,
Critical Reviews in Food Science and Nutrition
, 36S: 165-186, 1996 ). The offending proteins of many major sources of allergens have been characterized by clinical and molecular methods. The functions of allergenic proteins in vivo are diverse, ranging from enzymes to regulators of the cell cytoskeleton.
To understand the molecular basis of allergic disease, the important IgE binding epitopes of many allergen proteins have been mapped (Elsayed, S. and Apold, J.,
Allergy
38(7): 449-459, 1983; Elsayed, S. et al.,
Scand J. Clin. Lab. Invest. Suppl
. 204: 17-31 1991; Zhang, L., et al.,
Mol. Immunol
. 29(11): 1383-1389, 1992). The optimal peptide length for IgE binding has been reported to be between 8 and 12 amino acids. Conservation of epitope sequences is observed in homologous allergens of disparate species (Astwood, J. D., et al.,
Clin. Exp. Allergy
, 25: 66-72, 1995). Indeed, conservative substitutions introduced by site-directed mutagenesis reduce IgE binding of known epitopes when presented as peptides.
Food allergy occurs in 2-6% of the population. Eight foods or food groups (milk, eggs, fish, crustacea, wheat, peanuts, soybeans, and tree nuts) account for 90% of allergies to foods. Nevertheless, over 160 different foods have been reported to cause adverse reactions, including potato (Hefle, S., et al.,
Crit. Rev. in Food Sci. Nutr
., 36S: 69-90, 1996).
Mode of Action of Allergens
Regardless of the identity of the allergen, it is theorized that the underlying mechanism of allergen response is the same. Immediate hypersensitivity (or anaphylactic response) is a form of allergic reaction which develops very quickly, i.e., within seconds or minutes of exposure of the patient to the causative allergen, and is mediated by B lymphocyte IgE antibody production. Allergic patients exhibit elevated levels of IgE, mediating hypersensitivity by priming mast cells which are abundant in the skin, lymphoid organs, in the membranes of the eye, nose and mouth, and in the respiratory tree and intestines. The IgE in allergy-suffering patients becomes bound to the IgE receptors of mast cells. When this bound IgE is subsequently contacted by the appropriate allergen, the mast cell is caused to degranulate and release various substances such as histamine into the surrounding tissue (Church et al. In: Kay, A. B. ed.,
Allergy and Allergic Diseases
, Oxford, Blackwell Science, pp. 149-197, 1997).
It is the release of these substances which is responsible for the clinical symptoms typical of immediate hypersensitivity, namely contraction of smooth muscle in the airways or in the intestine, the dilation of small blood vessels, and the increase in their permeability to water and plasma proteins, the secretion of thick sticky mucus, and (in the skin) the stimulation of nerve endings that result in itching or pain. Immediate hypersensitivity is, at best, a nuisance to the suffer; at worst it can present very serious problems and can in rare cases even result in death.
Allergic Reactions to Potato
Food allergy to potato is considered rare in the general population (Castells, M. C., et al.,
Allergy Clin. Immunol
., 8: 1110-1114, 1986; Hannuksela, M., et al.,
Contact Dermatitis
, 3: 79-84, 1977; Golbert, T. M., et al.,
Journal of Allergy
, 44: 96-107, 1969). Approximately 200 individuals have participated in published clinical accounts of potato allergy (Hefle, S. et al.,
Critical Reviews in Food Science and Nutrition
, 36S: 69-90, 1996). A number of IgE binding proteins have been identified in potato tuber extracts (see Table 1), however the amino acid sequence and function of these proteins has not been determined (Wahl, R., et al.,
Intl. Arch. Allergy Appl. Immunol
., 92: 168-174, 1990).
TABLE 1
Studies of potato tuber IgE-binding proteins (allergens)
Study
Protein Characteristics
(Castells, M. C. et al.
Unknown 14 to 40 kDa
J. Allergy Clin. Immunol.
78, 1110-1114, 1986
(Wahl, R. et al. Int.
Unknown 42/43 kDa
Arch. Allergy Appl.
Immunol. 92:
Alibhai Murtaza F.
Astwood James D.
McWherter Charles A.
Sampson Hugh A.
Ball Timothy K.
Howrey Simon Arnold & White , LLP
Low Christopher S. F.
Mayes Laurie
Monsanto Technology LLC
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