# Patent

## Title
Preparation of deallergenized proteins and permuteins

## Abstract

Modified proteins are disclosed that maintain enzymatic and insecticidal activity while displaying reduced or eliminated allergenicity. Epitopes which bind to anti-patatin antibodies were identified, and removed via site directed mutagenesis. Tyrosines were observed to generally contribute to the allergenic properties of palatin proteins. Removal of glycosylation sites was observed to reduce or eliminate antibody binding. Permuteins are also disclosed which have a rearranged amino acid sequence while retaining enzymatic activity. Deallergenized proteins and permuteins can be used as insecticidal materials, as nutritional supplements, and as immunotherapeutic agents.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of co-pending U.S. patent application Ser. No. 09/755,630 filed Jan. 5, 2001 now U.S. Pat. No. 6,639,054, which claims the benefit of U.S. Provisional Application Ser. No. 60/174,669 filed on Jan. 6, 2000.


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 produciton. 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. J. Allergy Clin.
Unknown 14 to 40 kDa


Immunol. 78, 1110-1114, 1986)


(Wahl, R. et al. Int. Arch. Allergy Appl.
Unknown 42/43 kDa


Immunol. 92: 168-174, 1990)



Unknown 65 kDa



Unknown 26 kDa



Unknown 20 kDa



Unknown 14 kDa



Unknown <14 kDa ( ̃5 kDa)


(Ebner, C. et al. in: Wuthrich, B. &
Unknown 42/43 kDa


Ortolani, C. (eds.), Highlights in food


allergy. Monographs in Allergy,


Volume 32 Basil, Karger,


pp. 73-77, 1996)



Unknown 23 kDa



Unknown  ̃16 kDa



Unknown <14 kDa ( ̃5 kDa)









Improved Safety from the use of Hypoallergenic Proteins
Patatin has been identified as an allergenic protein (Seppala, U. et al., J. Allergy Clin. Immunol. 103:165-171, 1999). Accordingly, potato allergic subjects may not be able to safely consume products containing unmodified patatin protein, such as crops to which foliar applications of patatins have been applied, or crops which have been engineered to express patatin. In addition, proliferation of food allergens in the food supply is considered hazardous (Metcalfe, D. D., et al., Critical Reviews and Food Science and Nutrition, 36S: 165-186, 1996). There are additional concerns regarding the use of potentially allergenic food proteins where workers might be exposed to airborne particulates, initiating a new allergic response (Moneret-Vautrin, D. A., et al., Rev. Med. Interne., 17(7): 551-557, 1996).
Permuteins
Novel proteins generated by the method of sequence transposition resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J. Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem., 204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991; MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitro application of sequence rearrangement to proteins was described by Goldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion or sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain. This approach has been applied to proteins which range in size from 58 to 462 amino acids and represent a broad range of structural classes (Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983; Li and Coffino, Mol. Cell. Biol., 13: 2377-2383, 1993; Zhang, et al., Nature Struct. Biol., 1: 434-438, 1995; Buchwalder, et al., Biochemistry, 31: 1621-1630, 1994; Protasova, et al., Prot. Eng., 7: 1373-1377, 1995; Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533, 1994; Garrett, et al., Protein Science, 5: 204-211, 1996; Hahn, et al., Proc. Natl. Acad. Sci. USA., 91: 10417-10421, 1994; Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Luger, et al., Science, 243: 206-210, 1989; Luger, et al., Prot. Eng., 3: 249-258, 1990; Lin, et al., Protein Science, 4: 159-166, 1995; Vignais, et al., Protein Science, 4: 994-1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543-16551, 1995; Horlick, et al., Protein Eng., 5: 427-431, 1992; Kreitman, et al., Cytokine, 7: 311-318, 1995; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Koebnik and Kramer, J. Mol. Biol., 250: 617-626, 1995; Kreitman, et al., Proc. Natl. Acad. Sci., 91: 6889-6893, 1994).
There exists a need for the development of plant expressible insecticidal proteins which possess minimal or no allergenic properties.
SUMMARY OF THE INVENTION
Novel protein sequences, and nucleic acid sequences encoding them are disclosed. The proteins maintain desirable enzymatic and insecticidal properties while displaying reduced or eliminated allergenicity.
Allergenic epitopes are identified by scanning overlapping peptide sequences with an immunoreactivity assay. Alanine scanning and ‘rational substitution’ is performed on identified peptide sequences to determine specific amino acids which contribute to antibody binding, and presumably, to the allergenic properties of the whole protein. Individual mutations are introduced into the whole protein sequence by methods such as site directed mutagenesis of the encoding nucleic acid sequence to delete or modify the allergenic sequences.
Glycosylation target residues are identified within amino acid sequences of proteins which have demonstrated allergy eliciting properties. Glycosylation target amino acid residues are rationally substutited with other amino acid residues to eliminate glycosylation and to provide a variant deglycosylated protein. The variant protein may then exhibit reduced allergen eliciting properties and may also exhibit reduced binding to IgE within serum of patients observed to be allergic to said glycosylated protein.
Permuteins of the deallergenized protein sequences can be constructed to further reduce or eliminate allergic reactions. The encoding nucleic acid sequence is modified to produce a non-naturally occurring protein having a linear amino acid sequence different from the naturally occurring protein sequence, while maintaining enzymatic and insecticidal properties. The permutein is preferably produced in plant cells, and more preferably produced at a concentration which is toxic to insects ingesting the plant cells.
Methods for reducing, eliminating, or decreasing allergen eliciting properties of a protein are specifically contemplated herein. Such methods comprise steps including identifying one or more patients exhibiting an allergic sensitivity to an allergen eliciting protein and obtaining a sample of serum from the patient; exposing the patient serum to a first set of synthetic overlapping peptides which represent the allergen eliciting protein in order to identify such peptides which exhibit epitopes which bind to IgE present within the allergic patients' serum and wherein the IgE present in the serum has a specific affinity for the said allergen eliciting protein; producing a second set of peptides which are variant peptides based on the first set of peptides which were identified to bind specifically to IgE present in patient serum, wherein the second set variant peptides exhibit alanine scanning or rational scanning amino acid substitutions which exhibit reduced, decreased, or eliminated IgE binding when compared to the first set non-variant peptides, and wherein such substitutions which reduce, eliminate or decrease IgE binding are identified as result effective substitutions; and modifying the amino acid sequence of the allergen eliciting protein to contain one or more of said result effective substitutions, wherein the modified protein is a variant of the allergen eliciting protein which lacks allergen eliciting protein or exhibits reduced allergen eliciting properties, and wherein the variant of the allergen eliciting protein comprising one or more result effective substitutions exhibits reduced, decreased, or totally eliminated binding of IgE present within said patients' serum.
The novel proteins can be used in controlling insects, as nutritional supplements, in immunotherapy protocols, and in other potential applications. Transgenic plant cells and plants containing the encoding nucleic acid sequence can be particularly beneficial in the control of insects, and as a nutritional/immunotherapy material.



DESCRIPTION OF THE FIGURES
The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 illustrates the alignment of potato patatin PatA (acyl lipid hydrolase) with patatin (acyl lipid hydrolase) homologs and related amino acid sequences, the homologs and related sequences being from both dicot and monocot plant species.
FIG. 2 illustrates IgE binding to overlapping peptide sequences.
FIG. 3 illustrates construction of nucleic acid sequences encoding patatin permutein proteins, and in this figure for illustrative purposes a breakpoint at position 247 is shown.



DESCRIPTION OF THE SEQUENCE LISTINGS
The following description of the sequence listing forms part of the present specification and is included to further demonstrate certain aspects of the present invention. The invention can be better understood by reference to one or more of these sequences in combination with the detailed description of specific embodiments presented herein.













SEQ ID NO: 1
DNA sequence encoding a patatin (acyl lipid



hydrolase) protein


SEQ ID NO: 2
potato patatin protein sequence


SEQ ID NO: 3
thermal amplification primer


SEQ ID NO: 4
thermal amplification primer


SEQ ID NO: 5
thermal amplification product


SEQ ID NO: 6
Pre-cleavage patatin protein produced



in Pichia pastoris


SEQ ID NO: 7
Post-cleavage patatin protein produced in



Pichia pastoris


SEQ ID NO: 8
Y106F mutagenic primer


SEQ ID NO: 9
Y129F mutagenic primer


SEQ ID NO: 10
Y185F mutagenic primer


SEQ ID NO: 11
Y193F mutagenic primer


SEQ ID NO: 12
Y185F and Y193F mutagenic primer


SEQ ID NO: 13
Y270F mutagenic primer


SEQ ID NO: 14
Y316F mutagenic primer


SEQ ID NO: 15
Y362F mutagenic primer


SEQ ID NO: 16-104
Peptide scan sequences of a patatin protein


SEQ ID NO: 105-241
Alanine and rational scan sequences of



selected patatin peptides


SEQ ID NO: 242
thermal amplification primer 27


SEQ ID NO: 243
thermal amplification primer 48


SEQ ID NO: 244
thermal amplification primer 47


SEQ ID NO: 245
thermal amplification primer 36


SEQ ID NO: 246
pMON37402 sequence encoding permutein



protein


SEQ ID NO: 247
Permutein protein encoded from pMON37402



sequence


SEQ ID NO: 248
thermal amplification primer 58


SEQ ID NO: 249
thermal amplification primer 59


SEQ ID NO: 250
pMON37405 sequence encoding permutein



protein


SEQ ID NO: 251
Permutein protein encoded by pMON37405



sequence


SEQ ID NO: 252
thermal amplification primer 60


SEQ ID NO: 253
thermal amplification primer 61


SEQ ID NO: 254
pMON37406 sequence encoding permutein



protein


SEQ ID NO: 255
Permutein protein encoded by pMON37406



sequence


SEQ ID NO: 256
thermal amplification primer 62


SEQ ID NO: 257
thermal amplification primer 63


SEQ ID NO: 258
pMON37407 sequence encoding permutein



protein


SEQ ID NO: 259
Permutein protein encoded by pMON37407



sequence


SEQ ID NO: 260
thermal amplification primer 60


SEQ ID NO: 261
thermal amplification primer 65


SEQ ID NO: 262
pMON37408 sequence encoding permutein



protein


SEQ ID NO: 263
Permutein protein encoded by pMON37408



sequence


SEQ ID NO: 264
pMON40701 sequence encoding permutein



protein


SEQ ID NO: 265
Permutein protein encoded by pMON40701



sequence


SEQ ID NO: 266
thermal amplification primer Syn1


SEQ ID NO: 267
thermal amplification primer Syn2


SEQ ID NO: 268
thermal amplification primer Syn3


SEQ ID NO: 269
thermal amplification primer Syn4


SEQ ID NO: 270
pMON40703 sequence encoding permutein



protein


SEQ ID NO: 271
Permutein protein encoded by pMON40703



sequence


SEQ ID NO: 272
thermal amplification primer Syn10


SEQ ID NO: 273
thermal amplification primer Syn11


SEQ ID NO: 274
pMON40705 sequence encoding permutein



protein


SEQ ID NO: 275
Permutein protein encoded by pMON40705



sequence


SEQ ID NO: 276-277
Permutein linker sequences


SEQ ID NO: 278
Patatin isozyme PatA+ (including signal peptide)


SEQ ID NO: 279
Patatin isozyme PatB+ (including signal peptide)


SEQ ID NO: 280
Patatin isozyme PatFm (mature protein lacking



signal peptide)


SEQ ID NO: 281
Patatin isozyme PatIm (mature protein lacking



signal peptide)


SEQ ID NO: 282
Patatin isozyme PatL+ (including signal peptide)


SEQ ID NO: 283
Rational substitution peptide


SEQ ID NO: 284
Corn homolog peptide


SEQ ID NO: 285
patatin homolog Pat17 DNA coding sequence



and amino acid translation


SEQ ID NO: 286
patatin homolog Pat17 amino acid sequence


SEQ ID NO: 287
dicot patatin homolog amino acid sequence



pentin1_phb


SEQ ID NO: 288
dicot patatin homolog amino acid sequence



5c9_phb


SEQ ID NO: 289
maize patatin homolog amino acid sequence



corn1_pep


SEQ ID NO: 290
maize patatin homolog amino acid sequence



corn2_pep


SEQ ID NO: 291
maize patatin homolog amino acid sequence



corn3_pep


SEQ ID NO: 292
maize patatin homolog amino acid sequence



corn4_pep


SEQ ID NO: 293
maize patatin homolog amino acid sequence



corn5_pep









Definitions
The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention. Some words and phrases may also be defined in other sections of the specification. No limitation should be placed on the definitions presented for the terms below, where other meanings are evidenced elseqhere in the specification in addition to those specified below.
“Allergen” refers to a biological or chemical substance that induces an allergic reaction or response. An allergic response can be an immunoglobulin E-mediated response.
Amino acid codes: A (Ala)=alanine; C (Cys)=cysteine; D (Asp)=aspartic acid; E (Glu)=glutamic acid; F (Phe)=phenylalanine; G (Gly)=glycine; H (His)=histidine; I (Ile)=isoleucine; K (Lys)=lysine; L (Leu)=leucine; M (Met)=methionine; N (Asn)=asparagine; P (Pro)=proline; Q (Gln)=glutamine; R (Arg)=arginine; S (Ser)=serine; T (Thr)=threonine; V=(Val) valine; W (Trp)=tryptophan; Y (Tyr)=tyrosine.
“Amplification: refers to increasing the number of copies of a desired molecule.
“Coding sequence”, “open reading frame”, and “structural sequence” refer to the region of continuous sequential nucleic acid base pair triplets encoding a protein, polypeptide, or peptide sequence.
“Codon” refers to a sequence of three nucleotides that specify a particular amino acid.
“Complementarity” refers to the specific binding of adenine to thymine (or uracil in RNA) and cytosine to guanine on opposite strands of DNA or RNA.
“Deallergenize” (render hypoallergenic) refers to the method of engineering or modifying a protein or the encoding DNA such that the protein has a reduced or eliminated ability to induce an allergic response with respect to the ability of the unmodified protein. A deallergenized protein can be referred to as being hypoallergenic. The degree of deallergenization of a protein can be measured in vitro by the reduced binding of IgE antibodies.
“DNA segment heterologous to the promoter region” means that the coding DNA segment does not exist in nature in the same gene with the promoter to which it is now attached.
“DNA segment” refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species.
“Electroporation” refers to a method of introducing foreign DNA into cells that uses a brief, high voltage DC (direct current) charge to permeabilize the host cells, causing them to take up extra-chromosomal DNA.
“Encoding DNA” refers to chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA which encodes any of the enzymes discussed herein.
“Endogenous” refers to materials originating from within an organism or cell.
“Endonuclease” refers to an enzyme that hydrolyzes double stranded DNA at internal locations.
“Epitope” refers to a region on an allergen that interacts with the cells of the immune system. Epitopes are often further defined by the type of antibody or cell with which they interact, e.g. if the region reacts with B-cells or antibodies (IgE), it is called a B-cell epitope.
“Exogenous” refers to materials originating from outside of an organism or cell. This typically applies to nucleic acid molecules used in producing transformed or transgenic host cells and plants.
“Expressibly coupled” and “expressibly linked” refer to a promoter or promoter region and a coding or structural sequence in such an orientation and distance that transcription of the coding or structural sequence can be directed by the promoter or promoter region.
“Expression” refers to the transcription of a gene to produce the corresponding mRNA and translation of this mRNA to produce the corresponding gene product, i.e., a peptide, polypeptide, or protein.
“Heterologous DNA” refers to DNA from a source different than that of the recipient cell.
“Homologous DNA” refers to DNA from the same source as that of the recipient cell.
“Identity” refers to the degree of similarity between two nucleic acid or protein sequences. An alignment of the two sequences is performed by a suitable computer program. A widely used and accepted computer program for performing sequence alignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical. If the two compared sequences are of different lengths, the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
“IgE” (Immunoglobulin E) refers to a specific class of immunoglobulin secreted by B cells. IgE binds to specific receptors on Mast cells. Interaction of an allergen with mast cell-bound IgE may trigger allergic symptoms.
“Immunotherapy” refers to any type of treatment that targets the immune system. Allergy immunotherapy is a treatment in which a progressively increasing dose of an allergen is given in order to induce an immune response characterized by tolerance to the antigen/allergen, also known as desensitization.
“In vitro” refers to “in the laboratory” and/or “outside of a living organism”.
“In vivo” refers to “in a living organism”.
“Insecticidal polypeptide” refers to a polypeptide having insecticidal properties that adversely affects the growth and development of insect pests.
“Monocot” refers to plants having a single cotyledon (the first leaf of the embryo of seed plants); examples include cereals such as maize, rice, wheat, oats, and barley.
“Multiple cloning site” refers to an artificially constructed collection of restriction enzyme sites in a vector that facilitates insertion of foreign DNA into the vector.
“Mutation” refers to any change or alteration in a nucleic acid sequence. Several types exist, including point, frame shift, splicing, and insertion/deletions.
“Native” refers to “naturally occurring in the same organism”. For example, a native promoter is the promoter naturally found operatively linked to a given coding sequence in an organism. A native protein is one naturally found in nature and untouched or not otherwise manipulated by the hand of man.
“Nucleic acid segment” is a nucleic acid molecule that has been isolated free of total genomic DNA of a particular species, or that has been synthesized. Included with the term “nucleic acid segment” are DNA segments, recombinant vectors, plasmids, cosmids, phagemids, phage, viruses, etcetera.
“Nucleic acid” refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Nucleic acid codes: A=adenosine; C=cytosine; G=guanosine; T=thymidine; N=equimolar A, C, G, and T; I=deoxyinosine; K=equimolar G and T; R=equimolar A and G; S=equimolar C and G; W=equimolar A and T; Y=equimolar C and T.
“Open reading frame (ORF)” refers to a region of DNA or RNA encoding a peptide, polypeptide, or protein or capable of being translated to protein, or a regioiu of DNA capable of being transcribed into an RNA product.
“Plasmid” refers to a circular, extrachromosomal, self-replicating piece of DNA.
“Point mutation” refers to an alteration of a single nucleotide in a nucleic acid sequence.
“Polymerase chain reaction (PCR)” refers to an enzymatic technique to create multiple copies of one sequence of nucleic acid. Copies of DNA sequence are prepared by shuttling a DNA polymerase between two oligonucleotides. The basis of this amplification method is multiple cycles of temperature changes to denature, then re-anneal amplimers, followed by extension to synthesize new DNA strands in the region located between the flanking amplimers. Also known as thermal amplification.
“Probe” refers to a polynucleotide sequence which is complementary to a target polynucleotide sequence in the analyte. An antibody can also be used as a probe to detect the presence of an antigen. In that sense, the antigen binding domain of the antibody has some detectable affinity for the antigen and binds thereto. The binding of the antibody to the antigen can be measured by means known in the art, such as by chemiluminescence, phosphorescence, flourescence, colorimetric chemical deposition at the site of binding, or otherwise.
“Promoter” or “promoter region” refers to a DNA sequence, usually found upstream (5′) to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase and/or other factors necessary for start of transcription at the correct site. As contemplated herein, a promoter or promoter region includes variations of promoters derived by means of ligation to various regulatory sequences, random or controlled mutagenesis, and addition or duplication of enhancer sequences. The promoter region disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a host as part of a suitable recombinant vector, as demonstrated by its ability to produce mRNA.
“Recombinant DNA construct” or “recombinant vector” refers to any agent such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule in which one or more DNA sequences have been linked in a functionally operative manner. Such recombinant DNA constructs or vectors are capable of introducing a 5′ regulatory sequence or promoter region and a DNA sequence for a selected gene product into a cell in such a manner that the DNA sequence is transcribed into a functional mRNA which is translated and therefore expressed. Recombinant DNA constructs or recombinant vectors can be constructed to be capable of expressing antisense RNAs, in order to inhibit translation of a specific RNA of interest.
“Recombinant proteins”, also referred to as “heterologous proteins”, are proteins which are normally not produced by the host cell.
“Regeneration” refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant).
“Regeneration” refers to the process of growing a plant from a plant cell (e.g., plant protoplast or explant).
“Regulatory sequence” refers to a nucleotide sequence located upstream (5′), within, and/or downstream (3′) to a DNA sequence encoding a selected gene product whose transcription and expression is controlled by the regulatory sequence in conjunction with the protein synthetic apparatus of the cell.
“Restriction enzyme” refers to an enzyme that recognizes a specific palindromic sequence of nucleotides in double stranded DNA and cleaves both strands; also called a restriction endonuclease. Cleavage typically occurs within the restriction site.
“Result-effective substitution” (RES) refers to an amino acid substitution within an IgE-binding region (epitope) of a target protein which reduces or eliminates the IgE binding by that epitope. Examples herein are directed to patatin protein and homologues, however, as will be readily recognized by those skilled in the art, the method is more broadly applicable to proteins other than patatins, and in particular is applicable to any protein exhibiting allergen eliciting properties.
“Selectable marker” refers to a nucleic acid sequence whose expression confers a phenotype facilitating identification of cells containing the nucleic acid sequence. Selectable markers include those which confer resistance to toxic chemicals (e.g. ampicillin resistance, kanamycin resistance), complement a nutritional deficiency (e.g. uracil, histidine, leucine), or impart a visually distinguishing characteristic (e.g. color changes or fluorescence).
“Transcription” refers to the process of producing an RNA copy from a DNA template.
“Transformation” refers to a process of introducing an exogenous nucleic acid sequence (e.g., a vector, recombinant nucleic acid molecule) into a cell or protoplast in which that exogenous nucleic acid is incorporated into a chromosome or is capable of autonomous replication.
“Transformed cell” is a cell whose DNA has been altered by the introduction of an exogenous nucleic acid molecule into that cell.
“Transgenic cell” refers to any cell derived from or regenerated from a transformed cell or derived from a transgenic cell. Exemplary transgenic cells include plant calli derived from a transformed plant cell and particular cells such as leaf, root, stem, e.g., somatic cells, or reproductive (germ) cells obtained from a transgenic plant.
“Transgenic plant” refers to a plant or progeny thereof derived from a transformed plant cell or protoplast, wherein the plant DNA contains an introduced exogenous nucleic acid sequence not originally present in a native, non-transgenic plant of the same species. Alternatively, the plant DNA can contain the introduced nucleic acid sequence in a higher copy number than in the native, non-transgenic plant of the same species.
“Translation” refers to the production of protein from messenger RNA.
“Vector” refers to a plasmid, cosmid, bacteriophage, or virus that carries foreign DNA into a host organism.
“Western blot” refers to protein or proteins that have been separated by electrophoresis, transferred and immobilized onto a solid support, then probed with an antibody.
DETAILED DESCRIPTION OF THE INVENTION
Design of Deallergenized Patatin Proteins
Deallergenizing a protein can be accomplished by the identification of allergenic sites, followed by modification of the sites to reduce or eliminate the binding of antibodies to the sites. The IgE-binding regions of patatin were previously unreported. Mapping of the IgE epitopes was accomplished by synthesizing 10-mer peptides based on the patatin 17 protein sequence (SEQ ID NO: 2) which overlap by six amino acids. As potato proteins are denatured upon cooking potato products, it is expected that the 10-mer peptides sufficiently mimic the unfolded full length protein for antibody binding purposes. Peptides were identified based upon their ability to bind to IgE antibodies. Individual amino acids within the identified peptides were changed to reduce or eliminate binding to IgE present in sera from potato sensitive patients. These changes are termed result-effective amino acid substitutions (RES). The RES can be subsequently introduced into the full length protein by site directed mutagenesis of the encoding nucleic acid sequence or other means known in the art. Similar strategies have been employed elsewhere to determine the dominant IgE epitopes in a major peanut allergen (Stanley, J. S., et al., Arch. Biochem. Biophys., 342(2): 244-253, 1997).
Certain amino acid residues important for allergenicity of patatin are identified. Some of the designed patatin peptides wherein single amino acid residues were replaced with alanine or phenylalanine, showed significantly reduced or no binding to sera from potato sensitive patients.
A “deallergenized patatin” refers to a patatin protein differing in at least one of the amino acid residues as defined by the result effective substitutions resulting in the patatin protein having reduced reactivity towards sera from potato sensitive patients. The deallergenized patatin preferably maintains insecticidal properties, and preferably maintains its characteristic enzymatic profile.
Summary of Method to Deallergenize a Patatin Protein

    
    
        Mapping of IgE epitopes by immunoassay of synthetic overlapping peptides using sera from potato sensitive patients;
        Identification of result-effective substitutions by alanine scanning and/or rational scanning;
        Modification of the amino acid sequence of patatin by site-directed mutagenesis of the encoding nucleic acid sequence;
        Evaluation of enzyme activity (esterase) and/or insecticidal activity of the modified protein(s); and
        Evaluation of the new protein(s) for allergenicity by IgE immunoassay.
    
    


Nucleic acid sequences encoding patatin have been cloned by several investigators (e.g. Mignery, et al., Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, et al., Gene, 62: 27-44, 1988; WO 94/21805; Canadian Patent Application No. 2090552). These nucleic acid sequences can then be manipulated using site directed mutagenesis to encode a hypoallergenic patatin. These nucleic acid sequences can than be used to transform bacterial, yeast or plant cells, resulting in the production of hypoallergenic patatin protein.
Deallergenized Patatin Proteins
For simplicity, individual amino acids are referred to by their single letter codes. Correlation between the single letter codes, three letter codes, and full amino acid names is presented in the definitions section above.
One embodiment of the invention is an isolated deallergenized patatin protein. The protein is modified relative to the wild-type protein sequence such that they exhibit reduced binding to anti-patatin antibodies such as those obtained from humans or animals allergic to potatoes. The reduced binding is measured relative to the binding of the unmodified patatin protein to the anti-patatin antibodies.
The deallergenized patatin protein can comprise SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Preferably, the deallergenized patatin protein comprises SEQ ID NO:2 modified by one or more changes, or SEQ ID NO:7 modified by one or more changes. SEQ ID NO:7 differs from wild type SEQ ID NO:2 in that the first 22 amino acids of SEQ ID NO:2 are replaced with EAE (Glu-Ala-Glu). For example, the changes to SEQ ID NO:2 or SEQ ID NO:7 can be: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
More preferably, SEQ ID NO:2 is modified by the following changes or SEQ ID NO:7 is modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, SEQ ID NO:2 is modified by the following changes or SEQ ID NO:7 is modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Nucleic Acids
An additional embodiment of the invention is an isolated nucleic acid molecule segment comprising a structural nucleic acid sequence which encodes a deallergenized patatin protein.
The structural nucleic acid sequence can generally encode any deallergenized patatin protein. The structural nucleic acid sequence preferably encodes a deallergenized patatin protein comprising SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Alternatively, the structural nucleic acid sequence encodes SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
More preferably, the structural nucleic acid sequence encodes SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, the structural nucleic acid sequence encodes SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Recombinant Vectors
An additional embodiment is directed towards recombinant vectors comprising a structural nucleic acid sequence which encodes a deallergenized patatin protein. The recombinant vector comprises operatively linked in the 5′ to 3′ orientation: a promoter that directs transcription of a structural nucleic acid sequence; a structural nucleic acid sequence, and a 3′ transcription terminator.
The structural nucleic acid sequence can encode SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Alternatively, the recombinant vector comprises operatively linked in the 5′ to 3′ orientation: a promoter that directs transcription of a structural nucleic acid sequence; a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A; and a 3′ transcription terminator.
More preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Recombinant Host Cells
A further embodiment of the invention is directed towards recombinant host cells comprising a structural nucleic acid sequence encoding a deallergenized patatin protein. The recombinant host cell preferably produces a deallergenized patatin protein. More preferably, the recombinant host cell produces a deallergenized patatin protein in a concentration sufficient to inhibit growth or to kill an insect which ingests the recombinant host cell. The recombinant host cell can generally comprise any structural nucleic acid sequence encoding a deallergenized patatin protein.
The recombinant host cell can comprise a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Alternatively, the recombinant host cell comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
More preferably, the recombinant host cell comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, the recombinant host cell comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
The recombinant host cell can generally be any type of host cell, and preferably is a bacterial, fungal, or plant cell. The bacterial cell is preferably an Escherichia coli bacterial cell. The fungal cell is preferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plant cell can be a monocot, dicot, or conifer plant cell. The plant cell is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant cell. The recombinant host cell preferably further comprises operatively linked to the structural nucleic acid sequence a promoter that directs transcription of the structural nucleic acid sequence. The recombinant host cell preferably further comprises operatively linked to the structural nucleic acid sequence a 3′ transcription terminator and a polyadenylation site.
Recombinant Plants
An additional embodiment of the invention is a recombinant plant comprising a structural nucleic acid sequence encoding a deallergenized patatin protein. The recombinant plant preferably produces a deallergenized patatin protein. More preferably, the recombinant plant produces a deallergenized patatin protein in a concentration sufficient to inhibit growth or to kill an insect which ingests plant tissue from the recombinant plant.
The recombinant plant can comprise a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Alternatively, the recombinant plant can comprise a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
More preferably, the recombinant plant comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, the recombinant plant comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
The recombinant plant can generally be any type of plant. The plant can be a monocot, dicot, or conifer plant. The plant is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.
The recombinant plant preferably further comprises operatively linked to the structural nucleic acid sequence a promoter that directs transcription of the structural nucleic acid sequence. The recombinant plant preferably further comprises operatively linked to the structural nucleic acid sequence a 3′ transcription terminator and a polyadenylation site.
Methods of Preparation
Embodiments of the invention are further directed towards methods of preparing recombinant host cells and recombinant plants useful for the production of deallergenized patatin proteins.
A method of preparing a recombinant host cell useful for the production of deallergenized patatin proteins can comprise selecting a host cell; transforming the host cell with a recombinant vector; and obtaining recombinant host cells.
The recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Alternatively, the recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
More preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
The method can generally be used to prepare any type of recombinant host cell. Preferably, the method can be used to prepare a recombinant bacterial cell, a recombinant fungal cell, or a recombinant plant cell. The bacterial cell is preferably an Escherichia coli bacterial cell. The fungal cell is preferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plant cell can be a monocot, dicot, or conifer plant cell. The plant cell is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant cell.
An additional embodiment is directed towards methods for the preparation of recombinant plants useful for the production of deallergenized patatin proteins. The method can comprise selecting a host plant cell; transforming the host plant cell with a recombinant vector; obtaining recombinant host cells; and regenerating a recombinant plant from the recombinant host plant cells.
The recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified in one or more of the following regions, or SEQ ID NO:7 modified in one or more of the following regions. The single or multiple amino acid modifications reduce the binding of the modified protein relative to the binding of the corresponding unmodified protein. The regions for modification include amino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acid modifications include replacing an amino acid with A, E, F, P, or S. The modifications replace one or more amino acids in the identified regions, without increasing or decreasing the total number of amino acids in the protein.
Alternatively, the recombinant vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by one or more of the following changes or encoding SEQ ID NO:7 modified by one or more of the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the I corresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the S corresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the A corresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the T corresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the T corresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the K corresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the K corresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ ID NO:7 is replaced with E; the N corresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ ID NO:7 is replaced with A; the N corresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the K corresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ ID NO:7 is replaced with A; the R corresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ ID NO:7 is replaced with A; the F corresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ ID NO:7 is replaced with A; the K corresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ ID NO:7 is replaced with A; the L corresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the L corresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.
More preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
Most preferably, the vector comprises a structural nucleic acid sequence encoding SEQ ID NO:2 modified by the following changes or SEQ ID NO:7 modified by the following changes: the Y corresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Y corresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.
The recombinant plant can generally be any type of plant. The plant can be a monocot, dicot, or conifer plant. The plant is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.
Deallergenized patatin proteins can be prepared by isolating the deallergenized patatin protein from any one of the above described host cells or plants.
Deglycosylation
The examples herein provide evidence that glycosylation of can contribute to the allergenicity of a protein. Accordingly, rational substitution of amino acid residues likely to be the targets of glycosylation within a subject allergen protein may reduce or eliminate the allergenic properties of the protein without adversely affecting the enzymatic, insecticidal, antifungal or other functional properties of the protein.
Glycosylation commonly occurs as either N-linked or O-linked forms. N-linked glycosylation usually occurs at the motif Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro (Kasturi, L. et al., Biochem J. 323: 415-519, 1997; Melquist, J. L. et al., Biochemistry 37: 6833-6837, 1998). O-linked glycosylation occurs between the hydroxyl group of serine or threonine and an amino sugar.
Site directed mutagenesis of selected asparagine, serine, or threonine may be used to reduce or eliminate the glycosylation of patatin proteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motif reveals one occurrence at amino acid positions 202-204. Mutagenization of the nucleic acid sequence encoding this region may result in a reduced allergenicity of the encoded protein.
In order to test this conceptual approach to reducing allergenicity of patatin proteins, two sets of experiments were performed: a) production of patatin proteins in Escherichia coli, which do not glycosylate proteins; and b) production of patatin proteins with an N202Q site directed mutation.
Antibodies obtained from patients HS-07 and G15-MON (not potato allergic) did not show specific binding to wild type patatin, patatin produced in E. coli, or the N202Q variant. Antibodies obtained from patient HS-01 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-02 (potato allergic) bound strongly to wild type patatin, but extremely weakly to patatin produced in E. coli, and binding to the N202Q variant resembled vector controls. Antibodies obtained from patient HS-03 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-05 (potato allergic) bound to wild type patatin, but very weakly to patatin produced in E. coli and the N202Q variant. Antibodies obtained from patient HS-06 (potato allergic) strongly bound wild type patatin, the N202Q variant, and to patatin produced in E. coli. These results strongly suggest that glycosylation is at least partially responsible for the antigenic properties of patatin proteins, and that site directed mutagenesis may be used to reduce or eliminate specific antibody binding. Mutagenesis at position 202 of SEQ ID NO:2 may be useful for reducing or eliminating specific antibody binding.
Permuteins
The positions of the internal breakpoints described in the following Examples are found on the protein surface, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle. Breakpoints occurring below the protein surface can additionally be selected. The rearranged two subunits can be joined by a peptide linker. A preferred embodiment involves the linking of the N-terminal and C-terminal subunits by a three amino acid linker, although linkers of various sizes can be used. Additionally, the N-terminal and C-terminal subunits can be joined lacking a linker sequence. Furthermore, a portion of the C-terminal subunit can be deleted and the connection made from the truncated C-terminal subunit to the original N-terminal subunit and vice versa as previously described (Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984, 1993; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Protasova, et al., Prot. Eng., 7: 1373-1377, 1994).
The novel insecticidal proteins of the present invention can be represented by the formula:

X1-(L)a-X2

wherein;

    
    
        a is 0 or 1, and if a is 0, then the permutein does not contain a linker sequence;
        X1 is a polypeptide sequence corresponding to amino acids n+1 through J;
        X2 is a polypeptide corresponding to amino acids 1 through n;
        n is an integer ranging from 1 to J-1;
        J is an integer greater than n+1; and
        L is a linker.
    
    


In the formula above, the constituent amino acid residues of the novel insecticidal protein are numbered sequentially 1 through J from the original amino terminus to the original carboxyl terminus. A pair of adjacent amino acids within this protein can be numbered n and n+1 respectively where n is an integer ranging from 1 to J-1. The residue n+1 becomes the new N-terminus of the novel insecticidal protein and the residue n becomes the new C-terminus of the novel insecticidal protein.
For example, a parent protein sequence consisting of 120 amino acids can be selected as a starting point for designing a permutein (J=120). If the breakpoint is selected as being between position 40 and position 41, then n=40. If a linker is selected to join the two subunits, the resulting permutein will have the formula: (amino acids 41-120)-L-(amino acids 1-40). If a linker was not used, the resulting permutein will have the formula: (amino acids 41-120)-(amino acids 1-40).
The length of the amino acid sequence of the linker can be selected empirically, by using structural information, or by using a combination of the two approaches. When no structural information is available, a small series of linkers can be made whose length can span a range of 0 to 50 Å and whose sequence is chosen in order to be substantially consistent with surface exposure (Hopp and Woods, Mol. Immunol., 20: 483-489, 1983; Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982; Lee and Richards, J. Mol. Biol., 55: 379-400, 1971) and the ability to adopt a conformation which does not significantly affect the overall configuration of the protein (Karplus and Schulz, Naturwissenschaften, 72: 212-213, 1985). Assuming an average length of 2.0 to 3.8 Å per residue, this would mean the length to test would be between about 0 to about 30 residues, with 0 to about 15 residues being the preferred range. Accordingly, there are many such sequences that vary in length or composition that can serve as linkers with the primary consideration being that they be neither excessively long nor excessively short (Sandhu, et al., Critical Rev. Biotech., 12: 437-467, 1992). If the linker is too long, entropy effects may destabilize the three-dimensional fold and may affect protein folding. If the linker is too short, it may destabilize the molecule due to torsional or steric strain.
Use of the distance between the chain ends, defined as the distance between the C-alpha carbons, can be used to define the length of the sequence to be used, or at least to limit the number of possibilities that can be tested in an empirical selection of linkers. Using the calculated length as a guide, linkers with a range of number of residues (calculated using 2 to 3.8 Å per residue) can be selected. These linkers can be composed of the original sequence, shortened or lengthened as necessary, and when lengthened the additional residues can be chosen to be flexible and hydrophilic as described above; or optionally the original sequence can be substituted for using a series of linkers, one example being Gly-Pro-Gly (SEQ ID NO:277); or optionally a combination of the original sequence and new sequence having the appropriate total length can be used. An alternative short, flexible linker sequence is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276).
Selection of Permutein Breakpoints
Sequences of novel patatin analogs capable of folding to biologically active molecules can be prepared by appropriate selection of the beginning (amino terminus) and ending (carboxyl terminus) positions from within the original polypeptide chain while optionally using a linker sequence as described above. Amino and carboxyl termini can be selected from within a common stretch of sequence, referred to as a breakpoint region, using the guidelines described below. A novel amino acid sequence is thus generated by selecting amino and carboxyl termini from within the same breakpoint region. In many cases, the selection of the new termini will be such that the original position of the carboxyl terminus immediately preceded that of the amino terminus. However, selections of termini anywhere within the region may result in a functional protein, and that these will effectively lead to either deletions or additions to the amino or carboxyl portions of the new sequence.
The primary amino acid sequence of a protein dictates folding to the three-dimensional structure beneficial for expression of its biological function. It is possible to obtain and interpret three-dimensional structural information using x-ray diffraction of single protein crystals or nuclear magnetic resonance spectroscopy of protein solutions. Examples of structural information that are relevant to the identification of breakpoint regions include the location and type of protein secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets, chain reversals and turns, and loops (Kabsch and Sander, Biopolymers, 22: 2577-2637, 1983), the degree of solvent exposure of amino acid residues, the extent and type of interactions of residues with one another (Chothia, C., Ann. Rev. Biochem., 53: 537-572, 1984), and the static and dynamic distribution of conformations along the polypeptide chain (Alber and Mathews, Methods Enzymol., 154: 511-533, 1987). In some cases additional information is known about solvent exposure of residues, one example is a site of post-translational attachment of carbohydrate which is necessarily on the surface of the protein. When experimental structural information is not available, or when it is not feasible to obtain the information, methods are available to analyze the primary amino acid sequence in order to make predictions of protein secondary and tertiary structure, solvent accessibility and the occurrence of turns and loops (Fasman, G., Ed. Plenum, New York, 1989; Robson, B. and Gamier, J. Nature 361: 506, 1993).
Biochemical methods can be applicable for empirically determining surface exposure when direct structural methods are not feasible; for example, using the identification of sites of chain scission following limited proteolysis in order to infer surface exposure (Gentile, F. and Salvatore, G., Eur. J. Biochem., 218: 603-621, 1993). Thus, using either the experimentally derived structural information or predictive methods (Srinivasan, R. and Rose, G. D. Proteins, 22: 81-99, 1995), the parental amino acid sequence can be analyzed to classify regions according to whether or not they are integral to the maintenance of secondary and tertiary structure. The sequences within regions that are known to be involved in periodic secondary structure (alpha and 3-10 helices, parallel and anti-parallel beta sheets) are regions that should be avoided. Similarly, regions of amino acid sequence that are observed or predicted to have a low degree of solvent exposure are more likely to be part of the so-called hydrophobic core of the protein and should also be avoided for selection of amino and carboxyl termini. Regions that are known or predicted to be in surface turns or loops, and especially those regions that are known not to be required for biological activity, can be preferred sites for new amino and carboxyl termini. Stretches of amino acid sequence that are preferred based on the above criteria can be selected as breakpoint regions.
An embodiment of the invention is directed towards patatin permutein proteins. The permutein proteins preferably maintain esterase activity and insecticidal properties. The permutein proteins preferably are less allergenic than the wild type patatin protein to individuals or animals allergic to potatoes. This can be assayed by the binding of antibodies to the wild type patatin and patatin permutein proteins.
The permutein proteins can optionally contain a linker sequence. The linker can generally be any amino acid sequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) or Gly-Pro-Gly (SEQ ID NO:277), and more preferably is Gly-Pro-Gly (SEQ ID NO:277). Specific permutein proteins comprise: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQ ID NO:247, and SEQ ID NO:259.
Embodiments of the invention also include isolated nucleic acid molecule segments comprising a structural nucleic acid sequence encoding a patatin permutein protein. The encoded permutein protein can generally be any permutein protein, and preferably comprises (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQ ID NO:247, or SEQ ID NO:259. The linker can generally be any amino acid sequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) or Gly-Pro-Gly (SEQ ID NO:277), and more preferably is Gly-Pro-Gly (SEQ ID NO:277). Alternatively, the encoded patatin permutein protein can lack a linker sequence. The structural nucleic acid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258.
An embodiment of the invention is directed towards recombinant vectors which encode a patatin permutein protein. The vector can comprise operatively linked in the 5′ to 3′ orientation: a promoter that directs transcription of a structural nucleic acid sequence; a structural nucleic acid sequence encoding a protein selected from the group consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2); and a 3′ transcription terminator. The linker can comprise Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encoded patatin permutein protein can lack a linker sequence. The structural nucleic acid sequence can preferably be SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ ID NO:247 or SEQ ID NO:259.
An additional embodiment of the invention is directed towards recombinant host cells useful for the production of a patatin permutein protein. The recombinant host cell preferably produces a patatin permutein protein. More preferably, the recombinant host cell produces a patatin permutein protein in a concentration sufficient to inhibit growth or to kill an insect which ingests the recombinant host cell. The recombinant host cell can comprise a structural nucleic acid sequence encoding a protein selected from the group consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2). The linker can generally be any amino acid sequence, and preferably is Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encoded patatin permutein protein can lack a linker sequence. The structural nucleic acid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ ID NO:247 or SEQ ID NO:259. The structural nucleic acid sequence can be operatively linked to a promoter sequence that directs transcription of the structural nucleic acid sequence, a 3′ transcription terminator, and a 3′ polyadenylation signal sequence. The recombinant host cell can generally be any type of host cell, and preferably is a bacterial, fungal, or plant host cell. The bacterial cell is preferably an Escherichia coli bacterial cell. The fungal cell is preferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell. The plant cell can be a monocot, dicot, or conifer plant cell. The plant cell is preferably an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant cell.
An additional embodiment of the invention is directed towards recombinant plants which are useful for the production of patatin permutein proteins. The recombinant plant preferably produces a patatin permutein protein. More preferably, the recombinant plant produces a patatin permutein protein in a concentration sufficient to inhibit growth or to kill an insect which ingests tissue from the recombinant plant. The recombinant plant can comprise a structural nucleic acid sequence encoding a protein selected from the group consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2). The linker can comprise Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encoded protein can lack a linker sequence. The structural nucleic acid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ ID NO:247 or SEQ ID NO:259. The structural nucleic acid sequence can be operatively linked to a promoter sequence that directs transcription of the structural nucleic acid sequence, a 3′ transcription terminator, and a 3′ polyadenylation signal sequence. The recombinant plant can generally be any type of plant, and preferably is an alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.
Permutein proteins can be prepared by isolating the permutein protein from any one of the above described host cells or plants.
Immunotherapy for Potato Allergy
Immunotherapy for food allergy has been largely unsuccessful due to the lack of appropriate therapeutic reagents (Sampson, H. A., J. Allergy Clin. Immunol., 90(2): 151-152, 1992). Immunotherapy has typically involved the administration (orally or by subcutaneous injections) of increasing doses of crude protein extracts of the offending allergenic entities which usually contain variable mixes of many different proteins (Scheiner, O., Wien Klin Wochenschr., 105(22): 653-658, 1993). While there are reports of highly successful clinical applications of immunotherapy for food allergens (Romano, P. C., et al., Allergol. Immunopathol. (Madr), 12(4): 275-281, 1984), those reports are rare and the clinical literature in general recommends avoidance far more strongly than therapy (Gay, G., Allerg. Immunol. (Paris), 29(6): 169-170, 1997). One of the primary reasons for the failure of many clinical attempts to induce tolerance to allergens in general and food allergens in particular relates to anecdotal comments by numerous allergists, that patients don't tolerate the doses of allergen required to achieve tolerance. Animal studies examining the relationship of antigen dose and the induction of tolerance have demonstrated a strong positive correlation (Chen, Y., et al., Proc. Natl. Acad. Sci., U.S.A., 93: 388-391, 1996; Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758, 1997). Due to the very real possibility of inducing an anaphylactic reaction in patients with native allergen, most clinical therapists are quite hesitant to use high doses therapeutically and are therefore compromising the likelihood of successful therapy.
In recent reports, recombinant technology has been used to reduce the allergenic potential of a major allergen without modifying the T cell epitopes, and allowing higher doses of protein to be used in therapy (Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758, 1997). In addition, a lack of understanding about the appropriate route of administration, the uncertainty of mechanisms responsible for induction of allergy and the uncertainty of mechanisms by which immunotherapy suppresses or blocks the T cell-IgE-eosinophil/mast cell cycle have contributed to the large number of equivocal studies and clinical trials. Recent studies in animal models dealing with mechanisms, routes of administration, adjuvants and vaccine formulations have increased the likelihood that immunotherapy for allergies, including food allergies, will become a reproducibly successful clinical treatment when the appropriate therapeutic reagents are available (Sampson, H. A. and Burks, A. W., Annu. Rev. Nutr., 16: 161-177, 1996; Kaminogawa, S., Biosci. Biotechnol. Biochem., 60(11): 1749-1756, 1996; Chapman, M. D., et al., Allergy, 52: 374-379, 1997; Barbeau, W. E., Adv. Exp. Med. Biol., 415: 183-193, 1997; Cao, Y, et al., Immunology, 90(1): 46-51, 1997; Garside, P. and Mowat, A. M., Crit. Rev. Immunol., 17(2): 119-137, 1997; Rothe, M. J. and Grant-Kels, J. M., J. Am. Acad. Dermatol., 35(1): 1-13, 1996; Strobel, S., Allergy, 50(20): 18-25, 1995; Kruisbeek, A. M. and Amsen, D., Curr. Opin. Immunol., 8(2): 233-244, 1996; Herz, U., et al., Adv. Exp. Med. Biol., 409: 25-32, 1996; Litwin, A., et al., J. Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M. L., Mo. Med., 94(7): 311, 1997; Marshall, G. D., Jr. and Davis, F., Nat. Biotechnol., 15(8): 718-719, 1997; Van Deusen, M. A., et al., Ann. Allergy Asthma Immunol., 78: 573-580, 1997; Jacobsen, L., et al., Allergy, 52: 914-920, 1997, Scheiner, O. and Kraft, D., Allergy 50(5): 384-391, 1995).
Relative to immunotherapy, the critical aspects of the modified patatin genes described in this patent are that they can be used to synthesize purified, deallergenized-protein which can be used for patatin (potato) specific immunotherapy, with reduced potential for adverse and potentially fatal anaphylactic reactions in human or veterinary patients who have allergies to patatin or potatoes. Various strategies, including fixing or cross linking allergens, encapsulation of allergen for oral delivery, the use of small, T-cell epitope peptides and most recently, the use of engineered recombinant proteins, or modified gene vaccines are being tested in attempts to decrease the potential for anaphylactic reactions while inducing tolerance (Cao, Y., et al., Immunology, 90(1): 46-51, 1997; Chapman, M. D., et al., Allergy, 52: 374-379, 1997; Chapman, M. D., et al., Int. Arch. Allergy Immunol., 113(1-3): 102-104, 1997; Collins, S. P., et al., Clin. Exp. Allergy, 26(1): 36-42, 1996; Takai, T., et al., Mol. Immunol., 34(3): 255-261, 1997; Takai, T., et al., Nat. Biotechnol., 15(8) 754-758, 1997; Jirapongsananruk, O. and Leung, D. Y. M., Ann. Allergy Asthma Immunol., 79: 5-20, 1997; Litwin, A., et al., J. Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M. L., Mo. Med., 94(7): 311, 1997; Raz, E., et al., Proc. Natl. Acad. Sci., U.S.A., 93: 5141-5145, 1996; Hoyne, G. F., et al., Clin. Immunol. Immunopathol., 80: S23-30, 1996; Hoyne, G. F., et al., Int. Immunol., 9(8): 1165-1173, 1997; Vrtala, S., et al., J. Clin. Invest., 99(7): 1673-1681, 1997; Sato, Y., et al., Science, 273: 352-354, 1996; Lee, D. J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997; Tsitoura, D. C., et al., J. Immunol., 157(5): 2160-2165, 1996; Hsu, C. H., et al., Int. Immunol., 8(9):1405-1411, 1996; Hsu, C. H., et al., Nat. Med., 2(5): 540-544, 1996).
The instant invention uses an engineered patatin protein, as expressed in any living cell, with or without post-synthesis modifications, for immunotherapy by the routes of cutaneous or subcutaneous exposure, injection, or by oral, gastro-intestinal, respiratory or nasal application, either with, or without the use of specific carriers, vehicles and adjuvants. The direct application of nucleic acid encoding recombinant patatin as the in vivo (in the patient) expression template (gene) as RNA-, DNA- or gene-vaccines is also the intended use of the engineered genetic materials defined here, coding for patatin, but with modified IgE binding sites. It is also the intent of this patent to cover the use of these modified genes described here including insertion into various DNA vectors including adenovirus, retrovirus, pox virus and replicating or non-replicating eukaryotic expression plasmids (Lee, D. J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997) with various promoters and regulatory sequences, which can be inserted into the patient's somatic cells (dendritic cells, epithelial cells, muscle fiber-cells, fibroblasts, etc.) for the purpose of expressing the recombinant gene product to alter the patient's immune response to the patatin proteins (Lee D. J., et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997). Potential routes of administration foreseen in this application include previously described methods of encapsulation, emulsion, receptor or membrane fusion mediated uptake and methods of direct permeabilization or insertion of the DNA or corresponding RNA into the host cells.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLES
Example 1
Identification of Patatin as an Allergen
Since patatin is commonly obtained from an allergenic source (potato), it was hypothesized that patatins in fact encode an important class of offending potato allergens (patatin was reported as allergenic by Seppala, U. et al., J. Allergy Clin. Immunol. 103: 165-171, 1999). Assessment of potential allergens preferably include appropriate in vitro testing for IgE binding, in this case with potato allergic sera (Fuchs, R. L. and Astwood, J. D., Food Technology, 50: 83-88, 1996; Astwood, J. D., et al., 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). It is the recommendation of a working group organized by the IFBC and the ILSI Allergy and Immunology Institute that proteins encoded by nucleic acid sequences from allergenic sources such as potato (a “less-commonly” allergenic source) should be examined for their ability to react with IgEs of potato-allergic patients using a minimum of five individual patient sera (Metcalfe, D. D., et al., Critical Reviews in Food Science and Nutrition, 36S: 165-186, 1996). Patatin-17 protein was tested for IgE binding using standard in vitro testing with serum taken from patients with bona fide well defined clinically displayed potato allergy as described below.
Clinical Characterization of Potato Allergic Subjects (Serum Donors)
Patients who suffer from potato allergy were identified at Johns Hopkins Clinic (Baltimore, Md.) and were evaluated for potato allergy using clinical criteria outlined in Table 2.
Serum was obtained from patients with convincing clinical history of potato allergy. The convincing history was defined as being one or more of the following: a) positive potato allergic as evaluated by double-blind placebo-control food challenge b) anaphylaix and/or hospitalization due to the consumption of potatoes or c) dramatic skin test results.







TABLE 2







Clinical patient data












Flare/Wheal



Patient
Clinical History
(Skin prick test)
DBPCFC (potato)





HS01
Most recent hospitalization: Oct. 19, 1993
 7/19, 4/14, 7/17
Not performed



AD, A, AR, FH, MFS, IgE = 1397 KIAUa/L


HS02
Most recent hospitalization: Jun. 1994
20/26
Not performed



AD, FH, Latex (+) RAST, MFS, IgE = 7544 K/L


HS03
Most recent hospitalization: Jul. 27, 1995
 5/13
Yes



AD, A, FH, MFS, IgE = N/A


HS05
Most recent hospitalization May 30, 1995
 4/9
Yes



AD, A, FH, MFS, IgE = 12341 ng/ml


HS06
Most recent hospitalization Jun. 13, 1995
 5/20, 4/13, 5/12
Yes



AD, A FH, MFS IgE = N/A









HS07
Not potato allergic, allergic to egg, milk,
High IgE control serum, not



peanuts, seafood. AD, A, AR, FH, MFS
allergic to potato.


HS08
Non-atopic (normal)
Low IgE control serum





AD = Atopic dermatitis; 


FH = Food hypersensitivity; 


AR = Allergic rhinitis; 


A = Asthma; 


MFS = Multiple food sensitivity; 


N/A = not available. 






Example 2
Western Blotting of Patatin Proteins
Western blotting experiments were performed using patatin protein purified to near homogeneity from corn plants genetically engineered to produce patatin, patatin producing crude genetically engineered corn leaf extracts, crude potato tuber extracts, and non-transgenic corn leaf samples.
Protein samples were electrophoresed by SDS-PAGE (Laemmli, U. K., Nature 227: 680-685, 1970) and were electroblotted onto nitrocellulose. Protein blots were processed by standard Western blotting (immunoblotting) techniques and were incubated in potato allergic serum diluted 1:5 in PBS buffer for 1 hour. After washing the blots 3 times with PBS, the blots were incubated in biotinylated anti-IgE (Johns Hopkins Hospital, Baltimore, Md.) for 1 hour, followed by a 30 minute incubation in HRP-linked avidin (Promega, New York, N.Y.). IgE-reactive protein bands were visualized by DAB staining (3,3 diaminobenzidine). The blots were dried and photographed. Individual blots are labeled according to patient serum used. As a control, one blot was incubated in anti-IgE only.
Patatins were shown to be an allergen of potato by examining the reactivity of purified patatin to sera obtained from patients allergic to potato. Sera from five potato allergic subjects were tested by Western blotting techniques. All five sera reacted with purified patatin protein.
Patatin isozymes (SEQ ID NOS:278-282, FIG. 1) were tested for IgE binding by Western blotting. Isozymes of patatin were cloned into a yeast expression system and purified prior to analysis. The isozymes were subjected to IgE western blotting as described above with the exception that all five patient sera were pooled. The resulting Western blot of the yeast-expressed isozymes showed that all five isozymes bound IgE in a manner similar to patatin 17, and that all isozymes of patatin tested are also allergens.
Example 3
Western Blotting of Patatin Proteins
Eighty-nine 10-mer peptides were synthesized using the Genosys SPOTs system, each consecutive 10-mer overlapping by 6 amino acids based on the amino acid sequence of patatin 17 (SEQ ID NO:2). The peptides were evaluated for IgE binding with five different potato allergic patient sera using the same incubation procedures as described above. The results are summarized graphically in FIG. 2, showing major and minor allergenic epitopes. Interestingly, many of the immunogenic epitopes contain tyrosine. The peptide numbers, sequences, and immunoreactivity is detailed in Table 3.







TABLE 3







Peptide scan of patatin 17















Peptide #










(SEQ ID






Cumulative


NO)
Peptide Sequence
HS01
HS02
HS03
HS05
HS06
Total


















 1 (16)
QLGEMVTVLS
0.47
0.33
0.02
0.05
0.06
0.93



 2 (17)
MVTVLSIDGG
0.53
0.33
0.02
0.07
0.05
1


 3 (18)
LSIDGGGIRG
0.52
0.38
0.07
0.08
0.09
1.14


 4 (19)
GGGIRGIIPA
0.53
0.19
0.06
0.19
0.23
1.2


 5 (20)
RGIIPATILE
0.46
0.28
0.04
0.09
0.05
0.92


 6 (21)
PATILEFLEG
0.49
0.31
0.05
0.09
0.07
1.01


 7 (22)
LEFLEGQLQE
0.36
0.24
0.04
0.1
0.06
0.8


 8 (23)
EGQLQEMDNN
0.29
0.19
0.02
0.09
0.05
0.64


 9 (24)
QEMDNNADAR
0.22
0.13
0.01
0.05
0.04
0.45


10 (25)
NNADARLADY
0.21
0.17
0.03
0.05
0.07
0.53


11 (26)
ARLADYFDVI
0.54
0.31
0.16
0.15
0.25
1.41


12 (27)
DYFDVIGGTS
0.61
0.34
0.46
0.06
0.15
1.62


13 (28)
VIGGTSTGGL
0.63
0.72
0.05
0.15
0.09
1.64


14 (29)
TSTGGLLTAM
0.3
0.17
0.03
0.06
0.09
0.65


15 (30)
GLLTAMISTP
0.63
0.41
0.05
0.24
0.12
1.45


16 (31)
AMISTPNENN
0.34
0.18
0.02
0.07
0.02
0.63


17 (32)
TPNENNRPFA
0.46
0.22
0.03
0.19
0.07
0.97


18 (33)
NNRPFAAAKE
0.37
0.21
0.05
0.07
0.06
0.76


19 (34)
FAAAKEIVPF
0.52
0.29
0.08
0.11
0.08
1.08


20 (35)
KEIVPFYFEH
0.29
0.14
0.28
0.29
0.23
1.23


21 (36)
PFYFEHGPQI
0.65
0.06
1.08
0.51
0.17
2.47


22 (37)
EHGPQIFNPS
0.34
0.15
0.03
0.05
0.06
0.63


23 (38)
QIFNPSGQIL
0.33
0.29
0.02
0.07
0.07
0.78


24 (39)
PSGQILGPKY
0
0
0.02
0
0.05
0.07


25 (40)
ILGPKYDGKY
0
0
0.07
0
0.02
0.09


26 (41)
KYDGKYLMQV
0.02
0
0.11
0.01
0.04
0.18


27 (42)
KYLMQVLQEK
0.12
0.04
1.08
0.07
0.79
2.1


28 (43)
QVLQEKLGET
0.46
0.16
0.01
0.07
0.02
0.72


29 (44)
EKLGETRVHQ
0.5
0.12
0.01
0.07
0.04
0.74


30 (45)
ETRVHQALTE
0.42
0.16
0.03
0.05
0.03
0.69


31 (46)
HQALTEVVIS
0.43
0.21
0.04
0.1
0.05
0.83


32 (47)
TEVVISSFDI
0.44
0.25
0.05
0.08
0.04
0.86


33 (48)
ISSFDlKTNK
0.1
0.02
0.04
0.06
0.13
0.35


34 (49)
DIKTNKPVIF
0.57
0.22
0.04
0.18
0.28
1.29


35 (50)
NKPVIFTKSN
0
0.01
0.02
0.07
0.24
0.34


36 (51)
IFTKSNLANS
0
0
0.03
0.06
0.17
0.26


37 (52)
SNLANSPELD
0.43
0.96
0.01
0.09
0.02
1.51


38 (53)
NSPELDAKMY
0.18
0.12
0.01
0.05
0.05
0.41


39 (54)
LDAKMYDISY
0.54
0.26
0.19
0.15
0.23
1.37


40 (55)
MYDISYSTAA
0.92
0.08
0.52
0.04
0.22
1.78


41 (56)
SYSTAAAPTY
1.15
0.25
1.04
0.33
0.55
3.32


42 (57)
AAAPTYFPPH
1.02
0.52
1.12
0.81
0.86
4.33


43 (58)
TYFPPHYFVT
0.02
0.01
0.54
0.03
0.24
0.84


44 (59)
PHYFVTNTSN
0.03
0.01
1.17
0.13
0.44
1.78


45 (60)
VTNTSNGDEY
0.23
0.15
0.04
0.03
0.03
0.48


46 (61)
SNGDEYEFNL
0.33
0.25
0.08
0.1
0.11
0.87


47 (62)
EYEFNLVDGA
0.34
0.25
0.07
0.1
0.2
0.96


48 (63)
NLVDGAVATV
0.3
0.18
0.02
0.06
0.05
0.61


49 (64)
GAVATVADPA
0.45
0.54
0.01
0.07
0.02
1.09


50 (65)
TVADPALLSI
0.48
0.29
0.01
0.07
0.03
0.88


51 (66)
PALLSISVAT
0.65
0.33
0.02
0.1
0.01
1.11


52 (67)
SISVATRLAQ
0.61
0.23
0.14
0.53
0.53
2.04


53 (68)
ATRLAQKDPA
0.87
0.34
0.05
0.29
0.22
1.77


54 (69)
AQKDPAFASI
0.86
0.32
0.04
0.12
0.03
1.37


55 (70)
PAFASIRSLN
0.81
0.15
0.05
0.51
0.59
2.11


56 (71)
SIRSLNYKKM
0.07
0.01
0.17
0.07
0.11
0.43


57 (72)
LNYKKMLLLS
0.05
0.01
0.35
0.08
0.39
0.88


58 (73)
KMLLLSLGTG
1.15
0.15
0.04
0.38
0.71
2.43


59 (74)
LSLGTGTTSE
0.34
0.23
0.02
0.04
0.03
0.66


60 (75)
TGTTSEFDKT
0.92
0.39
0.6
0.1
0.09
2.1


61 (76)
SEFDKTYTAK
1.33
1.35
1.41
0.12
0.28
4.49


62 (77)
KTYTAKEAAT
1.36
0.94
1.11
0.76
0.4
4.57


63 (78)
AKEAATWTAV
0.45
0.15
0.01
0.2
0.04
0.85


64 (79)
ATWTAVHWML
0.1
0.02
0.01
0.08
0.06
0.27


65 (80)
AVHWMLVIQK
0.69
0.05
0.03
0.43
0.62
1.82


66 (81)
MLVIQKMTDA
0.32
0.15
0.02
0.15
0.03
0.67


67 (82)
QKMTDYYLST
0.26
0.125
0.03
0.21
0.05
0.675


68 (83)
DAASSYMTDY
0.2
0.14
0.08
0.08
0.1
0.6


69 (84)
SYMTDYYLST
0.5
0.03
0.32
0.06
0.11
1.02


70 (85)
DYYLSTAFQA
0.14
0
0.22
0.03
0.13
0.52


71 (86)
STAFQALDSK
0.4
0.3
0.04
0.06
0.08
0.88


72 (87)
QALDSKNNYL
0.44
0.46
0.28
0.26
0.43
1.87


73 (88)
SKNNYLRVQE
0.44
0.05
1.31
0.07
0.21
2.08


74 (89)
YLRVQENALT
1.38
0.03
1.31
0.11
0.2
3.03


75 (90)
QENALTGTTT
0.47
0.25
0
0.06
0
0.78


76 (91)
LTGTTTEMDD
0.41
0.24
0
0.06
0
0.71


77 (92)
TTEMDDASEA
0.38
0.3
0
0.05
0
0.73


78 (93)
DDASEANMEL
0.44
0.24
0
0.06
0
0.74


79 (94)
EANMELLVQV
0.42
0.27
0
0.04
0
0.73


80 (95)
ELLVQVGENL
0.4
0.25
0
0.05
0
0.7


81 (96)
QVGENLLKKP
0.44
0.14
0
0.07
0
0.65


82 (97)
NLLKKPVSED
0.47
0.2
0
0.03
0
0.7


83 (98)
KPVSEDNPET
0.27
0.21
0
0.03
0
0.51


84 (99)
EDNPETYEEA
0.13
0.11
0
0.01
0
0.25


85 (100)
ETYEEALKRF
1.26
1.2
1.36
0.53
0.71
5.06


86 (101)
EALKRFAKLL
1.38
0.04
0
1.06
1.12
3.6


87 (102)
RFAKLLSDRK
0.98
0.05
0
0.84
0.94
2.81


88 (103)
LLSDRKKLRA
0.2
0.01
0
0.37
0.51
1.09


89 (104)
RKKLRANKAS
0.28
0
0
0.31
0.64
1.23



Patient
41.84
20.565
18.1
14.17
16.55



Cumulative



Totals









Example 4
Identification of Result Effective Substitutions
For each major and several minor allergenic epitopes of patatin, result effective substitutions were identified by synthesizing peptides that were altered by individually substituting an alanine residue at each non-alanine position in the epitope. Similarly, the reported nucleic acid sequence encoding corn patatin (U.S. Pat. No. 5,882,668; clone 5c9) was evaluated for IgE binding by producing peptides at corresponding positions to the potato patatin protein.
For example, Epitope 41 was analyzed by alanine scanning and rational substitution as follows.










Epitope 41
SEFDKTYTAK
(SEQ ID NO:76)



Alanine scan
AEFDKTYTAK
(SEQ ID NO:165)



SAFDKTYTAK
(SEQ ID NO:166)



SEADKTYTAK
(SEQ ID NO:167)



SEFAKTYTAK
(SEQ ID NO:168)



SEFDATYTAK
(SEQ ID NO:169)



SEFDKAYTAK
(SEQ ID NO:170)



SEFDKTATAK
(SEQ ID NO:171)



SEFDKTYAAK
(SEQ ID NO:172)



SEFDKTYTAA
(SEQ ID NO:173)


Rational substitution
AFFDKTYTAK
(SEQ ID NO:283)



SEFDKTFTAK
(SEQ ID NO:176)


Corn homolog
CIFDSTYTAK
(SEQ ID NO:284)






Selected epitopes were analyzed by alanine scanning and rational substitution. Immunoassay with potato-allergic serum was used as described above. Table 4 summarizes the results of these experiments to identify result effective substitutions for patatin. Blank spaces in the table indicate that binding of the peptide to patient IgE was not detectable.








TABLE 4







Scanning of patatin for result



effective substitutions










Binding of modified




peptides by patient IgE



as measured by OD












Sequence
SEQ ID NO
HS03
HS06
HS01
HS02
















DYFDVIGGTS
105

0.12
0.16
0.36



DYFDVIAGTS
106

0.14
0.17
0.4


VIGGTSTGGL
107



0.04


VIAGTSTGAL
108


AFYFEHGPQI
109

0.96
0.5
0.78


PAYFEHGPQI
110

0.75
0.41
0.69


PFAFEHGPQI
111


PFYAEHGPQI
112

0.7
0.43
0.79


PFYFAHGPQI
113
0.93
1.07
0.59
1.44


PFYFEAGPQI
114
0.08
0.93
0.65
1.34


PFYFEHAPQI
115

0.75
0.54
1.11


PFYFEHGAQI
116

0.63
0.29
0.6


PFYFEHGPAI
117

0.63
0.25
0.56


PFYFEHGPQA
118

0.27
0.16
0.33


TFYLENGPKI
119
0.05
0.48
0.68
1.07


PFFFEHGPQI
120


AYLMQVLQEK
121

0.26
0.11
0.53


KALMQVLQEK
122


KYAMQVLQEK
123
0.11
0.43
0.1
1.25


KYLAQVLQEK
124
0.22
0.48
0.11
1.34


KYLMAVLQEK
125
0.22
0.83
0.16
1.33


KYLMQALQEK
126
0.11
0.6
0.15
0.95


KYLMQVAQEK
127

0.53
0.15
0.81


KYLMQVLAEK
128
0.06
0.69
0.11
1.34


KYLMQVLQAK
129
0.74
0.79
0.05
0.58


KYLMQVLQEA
130

0.28
0.27
0.37


VFLHDKIKSL
131
0.06
0.26

0.41


AYSTAAAPTY
132

0.1
0.12
0.12


SASTAAAPTY
133


SYATAAAPTY
134

0.16
0.13
0.37


SYSAAAAPTY
135

0.13
0.12
0.32


SYSTAAAATY
136

0.15
0.13
0.34


SYSTAAAPAY
137

0.15
0.14
0.29


SYSTAAAPTA
138

0.55
0.54
1.13


CISTSAAPTY
139
0.4


SYSTAAAPAF
140
0.39
1.02
0.65
1.42


AFAAAAAPTY
141


0.07


SYSTAAAPTF
142
0.15
0.97
0.48
1.09


STSAAPTYFP
143

0.21
0.23
0.39


STSAAPTFFP
144



0.23


STSAAPTAFP
145



0.08


STAAAPTFFP
146


0.12
0.28


AAAATYFPPH
147

0.13
0.1
0.05


AAAPAYFPPH
148


0.07
0.04


AAAPTAFPPH
149


AAAPTYAPPH
150

0.23
0.14
0.21


AAAPTYFAPH
151

0.45
0.18
0.44


AAAPTYFPAH
152

0.15
0.07
0.18


AAAPTYFPPA
153

0.1
0.06
0.31


SAAPTYFPAH
154

0.77
0.73
0.96


AAAPAFFPPH
155


AAAPPFFPPH
156


AAAPTFFPPH
157


SISVATRLAQ
158


0.26
0.26


AMSMLTKEVH
159


PAFASIRSLN
160


PNFNAGSPTE
161


KMLLLSLGTG
162


NYLIISVGTG
163
0.49
1.08
0.64
1.48


KMLLLSLGAG
164

0.13


AEFDKTYTAK
165
0.09
0.22

1.34


SAFDKTYTAK
166
0.66
0.71
0.06
1.42


SEADKTYTAK
167



0.99


SEFAKTYTAK
168
0.5
0.57

0.91


SEFDATYTAK
169



0.17


SEFDKAYTAK
170
0.1
0.24

1.38


SEFDKTATAK
171



0.81


SEFDKTYAAK
172
0.2
0.35

1.39


SEFDKTYTAA
173


0.1
1.18


KQAEKYTAEQ
174


0.08
0.24


SEFDAAFAAA
175


SEFDKTFTAK
176
0.09
0.16
0.07
1.45


AEKYTAEQCA
177


ATYTAKEAAT
178

0.24

0.18


KAYTAKEAAT
179

0.28

0.33


KTATAKEAAT
180


KTYAAKEAAT
181
0.1
0.32

0.73


KTYTAAEAAT
182



0.35


KTYTAKAAAT
183
0.4
0.59

0.82


KTYTAKEAAA
184



0.36


EKYTAEQCAK
185


AAFAAAEAAT
186


KTFTAKEAAT
187


QALHCEKKYL
188


QALDSKAAYL
189


QALDSKNNFL
190


QALHCENNFL
191


CEKKYLRIQD
192
1.01
0.16


SKNNFLRVQE
193


SENNYLRVQE
194
0.31
0.96
0.42
1


ALRVQENALT
195


YARVQENALT
196
1.06
1.02
0.05
0.54


YLAVQENALT
197
0.37
1.04
0.11
1.06


YLRAQENALT
198
1.1
1
0.06
1.26


YLRVAENALT
199
1.03
0.92
0.08
1.26


YLRVQANALT
200
1.05
0.92
0.06
1.24


YLRVQEAALT
201
0.93
0.92
0.07
1.11


YLRVQENAAT
202
0.94
0.93
0.04
1.24


YLRVQENALA
203
1.05
0.96
0.43
1.16


YLRIQDDTLT
204
1.07
0.85
0.39
1.12


YLTVAAAALT
205
1.05
0.86
0.28
1.33


FLRVQENALT
206


NNYLRVQENA
207
0.23
0.88
0.5
1.17


KKYLRIQDDT
208

0.26
0.09
0.37


NNFLRVQENA
209


NAYLRVQENA
210
0.17
1.02
0.53
1.06


ATYEEAKLRF
211
0.26
1.03

0.65


EAYEEALKRF
212
0.06
0.43

0.33


ETAEEALKRF
213

1.04


ETYAEALKRF
214
0.62
1.02

1.15


ETYEAALKRF
215
1.06
0.38

0.89


ETYEEAAKRF
216
0.08
0.1

0.9


ETYEEALARF
217



0.11


ETYEEALKAF
218



0.1


ETYEEALKRA
219



0.1


GTNAQSLADF
220


ETYEAALAAF
221
0.07
0.78
0.33
0.77


ETFEEALKRF
222


YEEALKTFAK
223
1.08
0.85
0.14
1.46


FEEALKRFAK
224
0.46
0.72

0.67


AALKRFAKLL
225
0.15
0.17


EAAKRFAKLL
226
0.08
0.33

0.05


EALARFAKLL
227

0.09


EALKAFAKLL
228


EALKRAAKLL
229
0.08
0.07


EALKRFAALL
230


EALKRFAKAL
231
0.06
0.09

0.1


EALKRFAKLA
232
0.06


0.1


QSLADFAKQL
233


AALAAFAKLL
234


LADFAKQLSD
235


DFAKQLSDER
236



0.17


AFAALLSDRK
237









Result effective substitutions were identified by a reduction in IgE binding ability with respect to the non-substituted peptide sequence. Table 5 shows the identified result effective substitutions. Blank spaces in the table indicate that binding of the peptide to patient IgE was not detectable. Many substitutions of alanine or phenylalanine for the original tyrosine resulted in reduced or eliminated antibody binding.







TABLE 5







Result effective substitutions of patatin













Location








(SEQ ID


NO)
Peptide (SEQ ID NO)
HS03
HS06
HS01
HS02


















Minor
PFYFEHGPQI

 (36)
1.08
0.17
0.65
0.06



Epitope
::A:::::::

(111)


21
::F:::::::
(r)
(120)



:::::::::A

(118)

0.27
0.16
0.33


Minor
KYLMQVLQEK

 (42)
1.08
0.79
0.12
0.04


Epitope
:A::::::::

(122)


27
:::::::::A

(130)

0.28
0.27
0.37



VFLHDKIKSL
(c)
(131)
0.06
0.26

0.41


Major
SYSTAAAPTY

 (56)
1.04
0.55
1.15
0.25


Epitope
A:::::::::

(132)

0.1
0.12
0.12


41
:A::::::::

(133)



AFAA::::::
(r)
(141)



0.007



CI::S:::::
(c)
(139)
0.04


Overlap
STAAAPTYFP

(238)


Epitope
::S::::A::
(r)
(145)



0.08


41/42


Major
AAAPTYFPPH

 (57)
1.12
0.86
1.02
0.52


Epitope
::::A:::::

(148)


0.07
0.04


42
:::::A::::

(149)


(57)
::::AF::::
(r)
(155)



::::PF::::
(r)
(156)



:::::F::::
(r)
(157)


Major
SEFDKTYTAK

 (76)
0.12
0.28
1.33
1.35


Epitope
::::A:::::

(169)



0.17


61
KQAE:YTAEQ
(c)
(174)


0.08
0.24



::::AAFA:A
(r)
(175)


Major
KTYTAKEAAT

 (77)
1.11
0.04
1.36
0.94


Epitope
A:::::::::

(178)

0.24

0.18


62
::A:::::::

(180)



:::::A::::

(182)



0.35



AAFA:A::::
(r)
(186)



::F:::::::
(r)
(187)



EK:::EQC:K
(c)
(185)


Minor
QALDSKNNYL

 (87)
0.28
0.43
0.44
0.46


Epitope
:::HCEKK::
(c)
(188)


72
::::::AA::
(r)
(189)



::::::::F:
(r)
(190)



:::::E::F:
(r)
(240)


Minor
SKNNYLRVQE

 (88)
1.31
0.21
0.44
0.05


epitope
::F:::::::
(r)
(193)


73


Minor
YLRVQENALT

 (87)
1.31
0.2
1.38
0.03


epitope
A:::::::::

(195)


74
F:::::::::
(r)
(206)


Overlap
NNYLRVQENA

(207)
0.23
0.88
0.5
1.17


epitope
::F:::::::
(r)
(209)


73/74


Major
ETYEEALKRF

(100)
1.36
0.71
1.26
1.2


epitope
:::::::A::

(217)



0.11


85
::::::::A:

(218)



0.1



:::::::::A

(219)



0.1



::F:::::::
(r)
(222)



G:NAQS:AD:
(c)
(220)


Major
EALKRFAKLL

(101)
0
1.12
1.38
0.04


Epitope
:::A::::::

(227)

0.09


86
::::A:::::

(228)



:::::A::::

(229)
0.08
0.07



:::::::A::

(230)



::::::::A:

(231)
0.06
0.09



:::::::::A

(232)
0.06



SD:AD:::Q:
(c)
(241)



A::AA:::::
(r)
(234)


Epitope
LKRFAKLLSD

(239)


overlap
(NO


86/87
BINDING)


Major
RFAKLLSDRK

(102)
0
0.94
0.98
0.05


Epitope
D:::Q:::ER
(c)
(236)



0.17


87
A::A::::::
(r)
(237)





(r) = rational; (c) = corn. 






Example 5
Site Directed Mutagenesis
To introduce site specific mutations, the cloned DNA sequence of patatin (SEQ ID NO:1 encoding patatin protein SEQ ID NO:2; pMON 26820) was subjected to PCR with primers SEQ ID NO:3 and SEQ ID NO:4 to incorporate part of the α-factor signal sequence (Pichia expression manual, Invitrogen, Carlsbad, Calif.), and EcoRI and XhoI restriction sites to facilitate cloning into the Pichia pastoris yeast secretion vector pPIC9 (GenBank accession number Z46233; Invitrogen, Carlsbad, Calif.). Typical PCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C. extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmol of each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl2, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, Calif.).
The amplified fragment SEQ ID NO:5 was digested with restriction enzymes XhoI and EcoRI and cloned into the pBluescript vector (Stratagene, La Jolla, Calif.), digested with the same two restriction enzymes. The resulting plasmid (pMON 26869) was used for oligonucleotide-directed mutagenesis using the Bio-Rad mutagenesis kit based on the method of Kunkel (Proc. Natl. Acad. Sci. U.S.A., 82: 477-492, 1985). Briefly, single-stranded pMON26869 was used as template for mutagenesis and was prepared by superinfection of plasmid containing cells with M13K07 (Gorman, et al., DNA Prot. Eng. Techniques, 2: 3-10, 1990). The mutagenic oligonucleotides are SEQ ID NOS:8-15 (reverse complement). DNA purified from transformed DH5αE. coli colonies was used for sequence determination. Sequencing was performed using the ABI PRISM sequencing kit (Perkin Elmer Biosystems, Foster City, Calif.). The resulting plasmid containing the mutation in the patatin gene was digested with restriction enzymes XhoI and EcoRI.
The patatin nucleic acid fragment was then ligated into the pPIC9 vector (Invitrogen, Carlsbad, Calif.), digested with the same two restriction enzymes to afford plasmid pMON37401. Pichia pastoris KM71 cells were electroporated with pMON37401 containing the appropriate mutation. The resulting transformed cells were used to produce protein in Pichia pastoris using the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.). The encoded protein contains an alpha factor signal cleavage site. Plasmid pMON37401 encodes SEQ ID NO:6 which is cleaved to afford SEQ ID NO:7, having four amino acids added at the N-terminus of amino acids 24-386 of SEQ ID NO:2. Position four of SEQ ID NO:7 therefore corresponds to position 23 of SEQ ID NO:2.
The concentration of patatin in the culture was determined using a patatin ELISA assay and the enzyme activity was measured using the method of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). The variants containing multiple mutations were further purified using Mono Q and hydrophobic interaction chromatography (HIC). Each culture was purified by first sizing on Amicon YM10 membranes (Millipore, Bedford, Mass.) to a >10 kDa fraction, followed by chromatography on the Mono Q HR 10/10 column (Pharmacia, Piscataway, N.J.). For chromatography on the Mono Q column, the samples were loaded on the column in 25 mM Tris pH 7.5 and eluted with a gradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatin protein were determined using SDS-PAGE. For chromatography on the HIC column, the appropriate fractions were pooled and dialyzed into 1 M ammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was then loaded on 16/10 phenyl Sepharose column (Pharmacia, Piscataway, N.J.) and eluted with a gradient of 25 mM Tris pH7.5.
The protein concentration was determined using the Bradford method, using BSA as a standard. SDS-PAGE analysis showed that these proteins were essentially pure. The esterase activity of the newly formed variants are shown in Table 6. The activity was determined using p-nitrophenyl caprate substrate as described by Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990).







TABLE 6







Esterase activity of patatin mutants









Activity


Variant
(mOD · min−1μg−1)











Wild type
93.2


Y106F
51.1


Y129F
74.7


Y185F
85.6


Y193F
82.2


Y185F/Y193F
99.4


Y270F
163.4


Y316F
94.88


Y362F
130.7


Y106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F
57.1


Y185F/Y193F/Y270F/Y316F/Y362F
161.5









Patatin proteins having a phenylalanine substitution at each of the amino acid positions 106, 129, 185, 193, 270, 316 and 362 (numbers correspond to positions in SEQ ID NO:2) of expressed SEQ ID NO:7 exhibit full enzyme activity. Proteins having multiple substitutions also displayed full enzyme activity.
In addition to nucleotide sequences encoding conservative amino acid changes within the fundamental polypeptide sequence, biologically functional equivalent nucleotide sequences include nucleotide sequences containing other base substitutions, additions, or deletions. These include nucleic acids containing the same inherent genetic information as that contained in the cDNA which encode peptides, polypeptides, or proteins conferring pathogen resistance the same as or similar to that of pathogen upon host cells and plants. Such nucleotide sequences can be referred to as “genetically equivalent modified forms” of the cDNA, and can be identified by the methods described herein.
Mutations made in the cDNA, plasmid DNA, genomic DNA, synthetic DNA, or other nucleic acid encoding the deallergenized gene preferably preserve the reading frame of the coding sequence. Furthermore, these mutations preferably do not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or hairpins, that would adversely affect mRNA translation.
Although mutation sites can be predetermined, it is not necessary that the nature of the mutations per se be predetermined. For example, in order to select for optimum characteristics of mutants at a given site, random mutagenesis can be conducted at the target codon.
Alternatively, mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native cDNA sequence. Following ligation, the resulting reconstructed nucleotide sequence encodes a derivative form having the desired amino acid insertion, substitution, or deletion.
Example 6
Construction of Permutein Sequences
Nucleic acid sequences encoding permutein proteins having rearranged N-terminus/C-terminus protein sequences can be made by following the general method described by Mullins et al. (J. Am. Chem. Soc. 116: 5529-5533, 1994). The steps are shown in FIG. 3. The Figure and the following Examples involve the design and use of a linker region separating the original C-terminus and N-terminus, but the use of a linker is not a critical or required element of permutein design.
Two sets of oligonucleotide primers are used in the construction of a nucleic acid sequence encoding a permutein protein. In the first step, oligonucleotide primers “new N-termini” and “linker start” are used in a PCR reaction to create amplified nucleic acid molecule “new N-termini fragment” that contains the nucleic acid sequence encoding the new N-terminal portion of the permutein protein, followed by the polypeptide linker that connects the C-terminal and N-terminal ends of the original protein. In the second step, oligonucleotide primers “new C-termini” and “linker end” are used in a PCR reaction to create amplified nucleic acid molecule “new C-termini fragment” that contains the nucleic acid sequence encoding the same linker as used above, followed by the new C-termini portion of the permutein protein. The “new N-termini” and “new C-termini” oligonucleotide primers are designed to include appropriate restriction enzyme recognition sites which assist in the cloning of the nucleic acid sequence encoding the permutein protein into plasmids.
Any suitable PCR conditions and polymerase can be used. It is desirable to use a thermostable DNA polymerase with high fidelity to reduce or eliminate the introduction of sequence errors. Typical PCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C. extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmol of each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl2, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, Calif.).
The amplified “new N-termini fragment” and “new C-termini fragment” are annealed to form a template in a third PCR reaction to amplify the full-length nucleic acid sequence encoding the permutein protein. The DNA fragments “new N-termini fragment” and “new C-termini fragment” are resolved on a 1% TAE gel, stained with ethidium bromide, and isolated using the QIAquick Gel Extraction Kit (Qiagen, Valencia, Calif.). These fragments are combined in equimolar quantities with oligonucleotide primers “new N-termini” and “new C-termini” in the third PCR reaction. The conditions for the PCR are the same as used previously. PCR reaction products can be purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.).
Alternatively, a linker sequence can be designed containing a restriction site, allowing direct ligation of the two amplified PCR products.
Example 7
Construction of Plasmid pMON 37402
The patatin protein contains a trypsin protease sensitive site at the arginine amino acid at position 246, as determined by electrophoresis of a trypsin digest reaction. In order to determine if the exposed protease site is an antigenic epitope, a permutein was constructed using positions 246-247 as a breakpoint.
The nucleic acid sequence encoding the permutein protein in plasmid pMON 37402 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “new N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 27 (SEQ ID NO:242) and 48 (SEQ ID NO:243). Nucleic acid molecule “new C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 36 (SEQ ID NO:245). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “new N-termini fragment” and “new C-termini fragment” using oligonucleotide primers 27 (SEQ ID NO:242) and 36 (SEQ ID NO:245).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON 37402 (containing SEQ ID NO:246, encoding protein sequence SEQ ID NO:247).
Example 8
Construction of Plasmid pMON 37405
Amino acids 201-202, near tyrosine 193, were chosen as a breakpoint for the construction of a permutein protein.
The nucleic acid sequence encoding the permutein protein in plasmid pMON 37405 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “New N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 58 (SEQ ID NO:249). Nucleic acid molecule “New C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 59 (SEQ ID NO:249). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “New N-termini fragment” and “New C-termini fragment” using oligonucleotide primers 58 (SEQ ID NO:248) and 59 (SEQ ID NO:249).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON 37405 (containing SEQ ID NO:250, encoding protein sequence SEQ ID NO:251).
Example 9
Construction of Plasmid pMON 37406
Amino acids 183-184, adjacent to tyrosine 185, were chosen as a breakpoint for the construction of a permutein protein.
The nucleic acid sequence encoding the permutein protein in plasmid pMON 37406 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “New N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 60 (SEQ ID NO:252). Nucleic acid molecule “New C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 61 (SEQ ID NO:253). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “New N-termini fragment” and “New C-termini fragment” using oligonucleotide primers 60 (SEQ ID NO:252) and 61 (SEQ ID NO:253).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON37406 (containing SEQ ID NO:254, encoding protein sequence SEQ ID NO:255).
Example 10
Construction of Plasmid pMON 37407
Amino acids 268-269, adjacent to tyrosine 270, were chosen as a breakpoint for the construction of a permutein protein.
The nucleic acid sequence encoding the permutein protein in plasmid pMON 37407 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “New N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 62 (SEQ ID NO:256). Nucleic acid molecule “New C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 63 (SEQ ID NO:257). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “New N-termini fragment” and “New C-termini fragment” using oligonucleotide primers 62 (SEQ ID NO:256) and 63 (SEQ ID NO:257).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON37407 (containing SEQ ID NO:258, encoding protein sequence SEQ ID NO:259).
Example 11
Construction of Plasmid pMON 37408
Amino acids 321-322, near tyrosine 216, were chosen as a breakpoint for the construction of a permutein protein.
The nucleic acid sequence encoding the permutein protein in plasmid pMON 37408 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “New N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243) and 64 (SEQ ID NO:260). Nucleic acid molecule “New C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 65 (SEQ ID NO:261). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “New N-termini fragment” and “New C-termini fragment” using oligonucleotide primers 64 (SEQ ID NO:260) and 65 (SEQ ID NO:261).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases XhoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON 26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digested with restriction endonucleases XhoI and EcoRI, and gel purified, resulting in an approximately 2900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON37408 (containing SEQ ID NO:262, encoding protein sequence SEQ ID NO:263).
Example 12
Production of Permutein Proteins in Pichia pastoris 
Plasmids pMON37402, pMON37405, pMON37406, pMON37407, and pMON37408 were individually used to electroporate KM71 cells from Pichia pastoris according to the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.). The resulting transformed cells were used to produce protein in Pichia pastoris following the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.).
The concentration of patatin in the culture was determined using a patatin ELISA assay and the enzyme activity was measured using the method of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). The variants containing multiple mutations were further purified using Mono Q and hydrophobic interaction chromatography (HIC). Each culture was purified by first sizing on YM10 membranes (Amicon, Mass.) to a [>10 kDa] fraction, followed by chromatography on the Mono Q HR 10/10 column (Pharmacia, N.J.). For chromatography on the Mono Q column, the samples were loaded on the column in 25 mM Tris pH 7.5 and eluted with a gradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatin protein were determined using SDS-PAGE. For chromatography on the HIC column, the appropriate fractions were pooled and dialyzed into 1 M ammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was then loaded on 16/10 phenyl Sepharose column (Pharmacia, N.J.) and eluted with a gradient of 25 mM Tris pH7.5.
The protein concentration was determined using the Bradford method, using BSA as a standard. SDS-PAGE analysis showed that these proteins were essentially pure. The esterase activity of the variants are shown in Table 7.







TABLE 7







Activity of permuteins











pMON
Breakpoint
Activity (ΔOD min−1μg−1)







Native enzyme

 83.21



pMON37402
246/247
66.7



pMON37405
201/202
No expression



pMON37406
183/184
No expression



pMON37407
268/269
12.1



pMON37408
321/322
No expression










The activity was determined using p-nitrophenyl caprate substrate as described by Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990).
Example 13
Insect Bioefficacy Assays
Assays for activity against larvae of SCRW are carried out by overlaying the test sample on an agar diet similar to that described by Marrone (J. Econ. Entom. 78: 290-293, 1985). Test samples were prepared in 25 mM Tris, pH 7.5 buffer. Neonate larvae are allowed to feed on the treated diet at 26° C., and mortality and growth stunting were evaluated after 5 or 6 days. The results of this assay are shown in Table 8.







TABLE 8







Insect bioefficacy assay









Protein (200 ppm)
Mean Survival Weight
% Weight Reduction





Tris buffer (control)
1.26 ± 0.3 
—


Wild Type
0.21 ± 0.02
83


pMON37402
0.21 ± 0.03
83


pMON37407
0.32 ± 0.04
75









These data demonstrate that the growth of the SCRW larvae is similarly reduced upon ingestion of the proteins encoded by pMON37402 and pMON37407 as compared to the wild type patatin protein.
Example 14
Permutein Sequences Improved for Monocot Expression
Modification of coding sequences has been demonstrated above to improve expression of insecticidal proteins. A modified coding sequence was thus designed to improve expression in plants, especially corn (SEQ ID NO:264).
Example 15
Construction of pMON40701 for Monocot Expression
Plasmid pMON19767 was digested with restriction endonucleases NcoI and EcoRI and the 1100 bp gene fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 was digested with restriction endonucleases NcoI and EcoRI, and gel purified, resulting in an approximately 3900 base pair vector fragment. The two purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON40700. Plasmid pMON40700 was digested with restriction endonuclease NotI and the resulting 2200 bp DNA fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested with restriction endonuclease NotI, and gel purified, resulting in an approximately 4200 base pair vector fragment. The two purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on kanamycin-containing plates. The resulting plasmid was designated pMON40701 (containing SEQ ID NO:264, encoding protein sequence SEQ ID NO:265).
Example 16
Construction of pMON40703 for Monocot Expression
The nucleic acid sequence encoding the permutein protein in plasmid pMON40703 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “New N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn1 (SEQ ID NO:266) and Syn2 (SEQ ID NO:267). Nucleic acid molecule “New C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn4 (SEQ ID NO:269). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “New N-termini fragment” and “New C-termini fragment” using oligonucleotide primers Syn1 (SEQ ID NO:266) and Syn4 (SEQ ID NO:269).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases NcoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 was digested with restriction endonucleases NcoI and EcoRI, and gel purified, resulting in an approximately 3900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON40702. Plasmid pMON40702 was digested with NotI, and the resulting 2200 bp DNA fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested with restriction endonuclease NotI, and gel purified, resulting in an approximately 4200 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on kanamycin-containing plates. The resulting plasmid was designated pMON40703 (containing SEQ ID NO:270, encoding protein sequence SEQ ID NO:271). Plasmid pMON40703 encodes a permutein protein with a “breakpoint” at positions 246/247 of the wild type patatin protein sequence (SEQ ID NO:2). The first 23 amino acids of SEQ ID NO:2 are a signal peptide sequence which is cleaved in the mature protein.
Example 17
Construction of pMON40705 for Monocot Expression
The nucleic acid sequence encoding the permutein protein in plasmid pMON40705 was created using the method illustrated in FIG. 3 and described in Example 6. Nucleic acid molecule “New N-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn10 (SEQ ID NO:272) and Syn2 (SEQ ID NO:267). Nucleic acid molecule “New C-termini fragment” was created and amplified from the sequence encoding patatin in plasmid pMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn11 (SEQ ID NO:273). The full-length nucleic acid molecule encoding the permutein protein was created and amplified from annealed fragments “New N-termini fragment” and “New C-termini fragment” using oligonucleotide primers Syn10 (SEQ ID NO:272) and Syn11 (SEQ ID NO:273).
The resulting amplified nucleic acid molecule was digested with restriction endonucleases NcoI and EcoRI, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON33719 was digested with restriction endonucleases NcoI and EcoRI, and gel purified, resulting in an approximately 3900 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on ampicillin-containing plates. Plasmid DNA was isolated and sequenced to confirm the presence of the correct insert. The resulting plasmid was designated pMON40704. Plasmid pMON40704 was digested with restriction endonuclease NotI, and the resulting 2200 bp DNA fragment was purified using the QIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested with restriction endonuclease NotI, and gel purified, resulting in an approximately 4200 base pair vector fragment. The purified restriction fragments were combined and ligated using T4 DNA ligase.
The ligation reaction mixture was used to transform E. coli strain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformant bacteria were selected on plates containing kanamycin. The resulting plasmid was designated pMON40705 (containing SEQ ID NO:274, encoding protein sequence SEQ ID NO:275). Plasmid pMON40705 encodes a permutein protein with a “breakpoint” at positions 268/269 of the wild type patatin protein sequence (SEQ ID NO:2). The first 23 amino acids of SEQ ID NO:2 are a signal peptide sequence which is cleaved in the mature protein.
Example 18
Transient Expression of Protein in Corn Leaf Protoplasts
Plasmids pMON40701, pMON40703, and pMON40705 (all containing the native signal sequence for vacuolar targeting) were separately electroporated into corn leaf protoplasts as described by Sheen (Plant Cell 3: 225-245, 1991). Protein was extracted with glass beads and the supernatant was assayed for protein expression using ELISA for patatin and NPTII. Expression of protein by the transformed corn protoplasts was confirmed by Western blot analysis. Expression results are shown in Table 9.







TABLE 9







ELISA data













Normalized





Expression



Patatin ELISA
NPTII ELISA
(Patatin ELISA/


Sample
(μg/mL)
(μg/mL)
NPTII ELISA)





pMON40701
1.1
0.6
1.8


pMON40703
2.1
0.3
7.0


pMON40705
1.3
0.6
2.2









The results indicate that the permutein encoded by plasmid pMON40703 surprisingly shows approximately 4-fold higher expression compared to the wild type enzyme.
Example 19
Deglycosylation of Protein Sequences
This example provides evidence that glycosylation of can contribute to the allergenicity of a protein. Accordingly, rational substitution of amino acid residues likely to be the targets of glycosylation within a subject allergen protein may reduce or eliminate the allergenic properties of the protein without adversely affecting the enzymatic, insecticidal, antifungal or other functional properties of the protein.
Glycosylation commonly occurs as either N-linked or O-linked forms. N-linked glycosylation usually occurs at the motif Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro (Kasturi, L. et al., Biochem J. 323: 415-519, 1997; Melquist, J. L. et al., Biochemistry 37: 6833-6837, 1998). O-linked glycosylation occurs between the hydroxyl group of serine or threonine and an amino sugar.
Site directed mutagenesis of selected asparagine, serine, or threonine may be used to reduce or eliminate the glycosylation of patatin proteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motif reveals one occurrence at amino acid positions 202-204. Mutagenization of the nucleic acid sequence encoding this region results in a reduced allergenicity of the encoded protein.
In order to test this approach to reducing allergenicity of patatin proteins, two sets of experiments were performed: a) production of patatin proteins in Escherichia coli, which do not glycosylate proteins; and b) production of patatin proteins with an N202Q site directed mutation.
Antibodies obtained from patients HS-07 and G15-MON (not potato allergic) did not show specific binding to wild type patatin, patatin produced in E. coli, or the N202Q variant. Antibodies obtained from patient HS-01 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-02 (potato allergic) bound strongly to wild type patatin, but extremely weakly to patatin produced in E. coli, and binding to the N202Q variant resembled vector controls. Antibodies obtained from patient HS-03 (potato allergic) bound to wild type patatin, but not to patatin produced in E. coli or the N202Q variant. Antibodies obtained from patient HS-05 (potato allergic) bound to wild type patatin, but very weakly to patatin produced in E. coli and the N202Q variant. Antibodies obtained from patient HS-06 (potato allergic) strongly bound wild type patatin, the N202Q variant, and to patatin produced in E. coli. These results strongly suggest that glycosylation is at least partially responsible for the antigenic properties of patatin proteins, and that site directed mutagenesis may be used to reduce or eliminate specific antibody binding. Mutagenesis at position 202 of SEQ ID NO:2 may be useful for reducing or eliminating specific antibody binding.
The deglycosylation approach was also tested using a patatin homolog, Pat17. As demonstrated above, patatin epitopes exhibiting IgE binding were identified, and each contained a Tyr residue. Substitution of these Tyr residues within each epitope led to loss of IgE binding. Site-directed mutagenesis was used to produce variants with individual and multiple Tyr substitutions in the protein, which was expressed in Pichia pastoris and assessed for enzyme activity. All the variants were found to have enzymatic activity no less than the wild type protein. A single variant with all 5 tyrosine residues substituted with phenylalinine was found to have insecticidal activity no less than the unsubstituted protein and was expressed in E. coli to produce the non-glycosylated version. The E. coli 5-“Tyr to Phe” variant was assessed for IgE binding. An isozyme of patatin, designated Pat17, was also expressed in corn to produce a plant glycoprotein and in E. coli to produce a nonglycosylated protein. Sera of seven patients (five exhibiting potato allergy and one exhibiting other allergies but no allergy to potatoes) were were used to assay Pat17 or Pat17 variant binding by immunoblot assay. Four of the five sera from patients exhibiting potato allergy showed either very weak or no binding to wild type patatin expressed in E. coli but did bind to the 5-Tyr variant. Serum from one patient exhibiting potato allergy showed strong binding to recombinant wild type patatin protein expressed in E. coli but weak binding to the 5-Tyr variant. Sera from all five patients exhibiting potato allergy bound strongly to patatin expressed in corn and native patatin present in potatoes. Serum from a control patient allergic to eggs, milk, peanuts and seafood, but exhibiting no allergy to potatoes showed no binding to patatin expressed in E. coli but did bind to patatin expressed in corn. Immunoblot results suggested that the sugar moiety in patatin is a non-specific IgE binding epitope and the polypeptide portion of patatin also contains immunogenic IgE epitopes.
Patients who suffer from potato allergy were identified at Johns Hopkins Clinic (Baltimore, Md.) and were evaluated for potato allergy using clinical criteria outlined in Table 2.
Serum was obtained from patients with convincing clinical history of potato allergy. The convincing history was defined as being one or more of the following: a) positive potato allergic reaction as evaluated by double-blind placebo-control food challenge b) anaphylaix and/or hospitalization due to the consumption of potatoes or c) dramatic skin test results.
Peptide Synthesis
Peptides were synthesized on cellulose membranes using the SPOTS system (Genosys Biotechnologies, TX). Membranes were stored at −20° C. until use.
Site Directed Mutagenesis
Site specific mutations were introduced into patatin by first incorporating part of the a-factor signal sequence (Pichia expression manual, Invitrogen, Carlsbad, Calif.) to the patatin gene using PCR. Primers used for the PCR were GGAGCTCGAGAAAAGAGAGGCTGAAGCTCAGTTGGGAGAAATGGTGACTGTTCT (SEQ ID NO: 3) (XhoI site in italics) and GGTCTAGAG GAATTCTCATTAATAAGAAG (SEQ ID NO: 4) (EcoRI site in italics). The primers contained restriction sites to facilitate cloning into Pichia pastoris yeast secretion vector pPIC9 (GenBank accession number Z46233; Invitrogen, Carlsbad, Calif.). Typical PCR conditions are 25 cycles 94° C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C. extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes. A 50 mL reaction contained 30 pmol of each primer and 1 mg of template DNA; and 1×PCR buffer with MgCl2, 200 mM dGTP, 200 mM dATP, 200 mM dTTP, 200 mM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, Calif.).
The amplified patatin gene was digested with restriction enzymes XhoI and EcoRI and cloned into the pBluescript vector (Stratagene, La Jolla, Calif.), digested with the same two restriction enzymes. The template plasmid DNA used for the PCR was pMON26820. The resulting plasmid (pMON 26869) was used for oligonucleotide-directed mutagenesis using the Bio-Rad mutagenesis kit based on the method of Kunkel et al., Proc Natl Acad Sci USA 82, 477-92 (1985). Briefly, single-stranded pMON26869 was used as template for mutagenesis and was prepared by superinfection of plasmid containing cells with M13K07 (Gorman et al., DNA and Protein Engineering techniques 2, 3-10 (1990)). DNA purified from transformed DH5a E. coli colonies was used for sequence determination. Sequencing was performed using the ABI PRISM sequencing kit (Perkin Elmer Biosystems, Foster City, Calif.).
Protein Expression in Pichia pastoris 
Plasmids containing the mutations in the patatin gene were digested with restriction enzymes XhoI and EcoRI. The patatin nucleic acid fragment was then ligated into the pPIC9 vector (Invitrogen, Carlsbad, Calif.), digested with the same two restriction enzymes to afford plasmid pMON37401. Pichia pastoris KM71 cells were electroporated with pMON37401 containing the appropriate mutation. The resulting transformed cells were used to produce protein in Pichia pastoris using the procedure supplied by the manufacturer (Invitrogen, Carlsbad, Calif.). The proteins were purified in the same way as the proteins expressed in E. coli (see below).
Western Blotting of Proteins
Protein samples were electrophoresed by SDS-PAGE and electroblotted onto PVDF membrane (Millipore, Bedford Mass.). Protein blots were processed by standard Western blotting (immunoblotting) techniques and were incubated in potato allergic serum diluted 1:5 in PBS buffer for 1 hour. After washing the blots 3 times with PBS, the blots were incubated in biotinylated anti-IgE (Johns Hopkins Hospital, Baltimore Md.) for 1 hour, followed by a 30 minute incubation in HRP-linked avidin (Promega, New York, N.Y.). IgE-reactive protein bands were visualized by using the ECL system (Amersham Pharmacia Biotech, N.J.). As a control, one blot was incubated in anti-IgE only. His-tagged glyphosate oxidase and potato extracts was prepared and provided for this study by Regulatory Sciences, Monsanto Company. The peptides were evaluated using the same incubation procedures as described above.
Expression and Purification of Patatin in Corn
An isozyme of patatin, Pat17, was generated for expression in corn using a modified plant optimized gene sequence as described by Brown et al (U.S. Pat. No. 5,689,052). All the constructs contained the native 23 amino acid signal peptide for vacuolar targeting. Corn was transformed by microprojectile bombardment (Morrish et al., in Transgenic plants. Fundamentals and Applications (ed. Hiatt, A.) 133-171 (Marcel Dekker, New York, 1993); Songstad et al., In Vitro Cell Dev Biol—Plant 32, 179-183 (1996)). Protein from the transformed corn plants was purified by first grinding the leaves in liquid nitrogen and extracting the protein using 25 mM Tris/HCl. The plant extract was further dialyzed against 25 mM Tris/HCl pH 7.5. The plant extract was then loaded onto Mono Q HR 10/10 anion-exchange column (Amersham Pharmacia, N.J.) equilibrated with 25 mM Tris/HCl pH 7.5 (buffer A). The protein was eluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) using a linear gradient of 0-100% buffer B using an HPLC system (Shimadzu). Fractions containing protein were assayed for esterase activity and dialyzed against 25 mM Tris/HCl pH 7.5, 1 M Ammonium Sulfate (buffer C). The protein was purified to homogeneity by loading onto a phenyl-Sepharose 16/10 column (Amersham Pharmacia, N.J.) equilibrated with buffer C. Esterase active fractions were pooled and dialyzed against 25 mM Tris/HCl pH 7.5.
Expression and Purification of Patatin in E. coli 
Pat17 was expressed in E. coli using the pET expression system (Novagen, Wis.). The coding region of the mature Pat17 gene (without its signal peptide) was amplified by PCR using the primers 5′-GGGCCATGGCGCAGTTGGGAGAAATGGTG-3′ (SEQ ID NO: 294) (NcoI site in italics) and 5′-AACAAAGCTTCTTATTGAGGTGCGGCCGCTTGCATGC-3′ (SEQ ID NO: 295) (NotI site in italics) using standard PCR reaction conditions as described in the Gene Amp kit (Perkin-Elmer Cetus, Conn.) and an annealing temperature of 40° C. The template was plasmid pMON26820. The resulting DNA was digested with NcoI and NotI and cloned into a modified pET24d plasmid, designed to add an N-terminal hexa-histidine tag to the protein. The correct sequence of the PCR product was verified by sequencing, and the plasmid was transformed into E.coli BL21 (DE3), and transformants selected on LB containing 25 mg/mL kanamycin. The expression strain was grown in LB containing 25 mg/mL kanamycin and induced for 8 hrs at 28° C. with 1 mM IPTG. Cells were harvested and washed in 50 mM Tris/HCl pH 8.5, 150 mM NaCI, and lysed by French Press at 20,000 psi. His-tagged protein was recovered in the soluble fraction of lysed cells and subsequently purified using Ni—NTA resin as described in the QIAexpressionist manual (Qiagen Calif.). The partially purified protein was then dialyzed against 25 mM Tris/HCl pH 7.5 (buffer A) and loaded onto Mono Q HR 10/10 anion-exchange column (Amershani Pharmacia, N.J.) equilibrated with buffer A. The protein was eluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) using a linear gradient of 0-100% buffer B run over 30 min at a flow rate of 4 mL/min using an HPLC system (Shimadzu). Fractions containing protein were assayed for esterase activity. Esterase active fractions were pooled, concentrated and dialyzed against 25 mM Tris/HCl pH 7.5 and stored at 4° C.
Enzyme Activity Assays
Enzyme activity was measured as described previously using p-nitrophenyl caprate (Sigma, MO) as a substrate, dissolved in dimethylsulfoxide (5 mM stock solution) and diluted in 4% Triton X-100, 1% SDS to a final concentration of 1 mM. For the assay, 20 mL of protein solution was added to a mixture of 25 mL of the 1 mM substrate solution and 80 mL of 50 mM Tris pH 8.5. The enzyme activity was monitored at 405 nm in 6 sec interval for a period of 10 min. Esterase activity was expressed as DOD min−1 mg−1 protein.
Insect Bioassay
The protein was also assayed for activity against larvae of Diabrotica virigifera (Western corn rootworm) by overlaying the test sample on an agar diet similar to that described previously (Marrone et al., J. Econ. Entom. 78, 290-3 (1985)). Proteins to be tested were diluted in 25 mM Tris/HCl pH 7.5 and overlayed on the diet surface. Neonate larvae were allowed to feed on the diet and mortality and growth stunting were evaluated after 6 days.
IgE Binding Epitopes on Patatin
A panel of eighty-nine overlapping peptides representing the amino acid sequence of patatin were synthesized to determine the regions responsible for IgE binding. Each peptide was 10 amino acids long and consisted of 6 amino acid overlap between the consecutive peptides. The peptides were evaluated for IgE binding with five different potato allergic patient sera. Patatin has 3 major epitopes. These major IgE binding regions represent amino acids 184-193, 188-197, 269-278 and 360-369. Other minor IgE binding regions represent amino acids 104-113, 138-147 and 316-325. The amino acids essential for IgE binding in each major and minor epitopes were determined by synthesizing peptides with single amino acid changes at each position by individually substituting an alanine residue at each non-alanine position in the epitopes. The resulting alanine substituted peptides were evaluated for IgE binding. Result effective substitutions were identified by a reduction in IgE binding with respect to the non-substituted peptide sequence. It was very interesting to note that all the epitopes contained a Tyr residue and substitution of this Tyr for Ala or Phe eliminated IgE binding.
Enzyme and Bioactivity
The Tyr residues identified to be critical for IgE binding in each of the epitopes were substituted with Phe either individually or in concert using site-directed mutagenesis. All the variants were expressed in Pichia pastoris and assessed for enzyme activity and insecticidal activity. The variants included Y106F, Y129F, Y185F, Y193F, Y270F, Y316F, Y362F, Y185F/Y193F, Y185F/Y193F/Y270F/Y316F/Y362F (5-Tyr) and Y106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F (7-Tyr). All the variants maintained enzyme activity. The 5-Tyr and 7-Tyr variants were then assessed for insecticidal activity by overlaying protein (200 ppm final concentration). The proteins caused significant stunting of the larval growth as measured by the weight of the larvae after 6 days with the 5-Tyr variant showing higher insecticidal activity compared to the 7-Tyr and wild type proteins. The 7-Tyr variant was unstable upon long term storage at 4° C. and thus was not pursued further.
Immunoblotting
In order to test if the glycan moiety on patatin was important for binding of IgE, Pat17 was expressed in E. coli to produce a nonglycosylated protein and in corn to produce a plant glycosylated protein. The 5-Tyr variant was also expressed in E. coli to assess the individual contribution of the linear epitopes without the glycan moiety on the protein. The proteins were tested for binding to IgE using sera from five patients with allergy to potatoes and sera from one patient with allergies to many things but no allergy to potatoes. Proteins from both corn and E. coli were purified to homogeneity. These proteins were transferred to PVDF membrane (Millipore, Mass.) and subsequently probed with sera from patients with and without allergy to potatoes. A His-tagged glyphosate oxidase control was included in all the studies to verify that the His-tag did not affect the binding of IgE. Serum obtained from patient HS-07 (no allergy to potatoes) did not bind Pat17 expressed in E. coli but showed good binding to Pat17 from corn and also a protein at the same molecular weight in potato extract. It is interesting to note that this sera also showed strong binding to another protein (>46 kDa) in the potato. Sera from patients HS-01, HS-02, HS-03, HS-05 (allergy to potatoes) shows strong binding to Pat17 expressed in corn, but very weak to no binding to Pat17 produced in E. coli. Also, the sera from patients HS-01, HS-2, HS-03 and HS-05 bound to a protein of similar molecular weight in the potato extract. Sera from patients HS-01, HS-02 and HS-03 also showed binding to another protein in potato extract of a lower molecular weight (<30 kDa). Serum obtained from patient HS-06 (allergic to potatoes) showed very strong binding to wild type patatin expressed in both corn and E. coli but weaker binding to the 5-Tyr variant expressed in E. coli. Sera from HS-06 also showed very strong binding to a protein in potato extract with similar molecular weight as patatin. The sera from all the patients showed no binding to His-tagged glyphosate oxidase indicating that the His-tag does not bind IgE. These results strongly suggest that the glycan moiety on Pat17 is responsible for IgE binding in some potato allergic patients and linear epitopes also contribute to the antigenicity of patatin.
Example 20
Alternative Nucleic Acid and Protein Sequences
For future variations of the patatin protein, sequences showing high similarity to the sequences disclosed herein could be used in producing deallergenized patatin proteins and permuteins. For example, a BLAST search (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990) can be performed to identify additional patatin sequences. Sources other than those disclosed herein can be used to obtain a patatin nucleic acid sequence, and the encoded patatin protein. Furthermore, subunit sequences from different organisms can be combined to create a novel patatin sequence incorporating structural, regulatory, and enzymatic properties from different sources.
Example 21
Nucleic Acid Mutation and Hybridization
Variations in the nucleic acid sequence encoding a patatin protein may lead to mutant patatin protein sequences that display equivalent or superior enzymatic characteristics when compared to the sequences disclosed herein. This invention accordingly encompasses nucleic acid sequences which are similar to the sequences disclosed herein, protein sequences which are similar to the sequences disclosed herein, and the nucleic acid sequences that encode them. Mutations can include deletions, insertions, truncations, substitutions, fusions, shuffling of subunit sequences, and the like.
Mutations to a nucleic acid sequence can be introduced in either a specific or random manner, both of which are well known to those of skill in the art of molecular biology. A myriad of site-directed mutagenesis techniques exist, typically using oligonucleotides to introduce mutations at specific locations in a nucleic acid sequence. Examples include single strand rescue (Kunkel, T. Proc. Natl. Acad. Sci. U.S.A., 82: 488-492, 1985), unique site elimination (Deng and Nickloff, Anal. Biochem. 200: 81, 1992), nick protection (Vandeyar, et al. Gene 65: 129-133, 1988), and PCR (Costa, et al. Methods Mol. Biol. 57: 31-44, 1996). Random or non-specific mutations can be generated by chemical agents (for a general review, see Singer and Kusmierek, Ann. Rev. Biochem. 52: 655-693, 1982) such as nitrosoguanidine (Cerda-Olmedo et al., J. Mol. Biol. 33: 705-719, 1968; Guerola, et al. Nature New Biol. 230: 122-125, 1971) and 2-aminopurine (Rogan and Bessman, J. Bacteriol. 103: 622-633, 1970), or by biological methods such as passage through mutator strains (Greener et al. Mol. Biotechnol. 7: 189-195, 1997).
Nucleic acid hybridization is a technique well known to those of skill in the art of DNA manipulation. The hybridization properties of a given pair of nucleic acids is an indication of their similarity or identity. Mutated nucleic acid sequences can be selected for their similarity to the disclosed patatin nucleic acid sequences on the basis of their hybridization to the disclosed sequences. Low stringency conditions can be used to select sequences with multiple mutations. One may wish to employ conditions such as about 0.15 M to about 0.9 M sodium chloride, at temperatures ranging from about 20° C. to about 55° C. High stringency conditions can be used to select for nucleic acid sequences with higher degrees of identity to the disclosed sequences. Conditions employed may include about 0.02 M to about 0.15 M sodium chloride, about 0.5% to about 5% casein, about 0.02% SDS and/or about 0.1% N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, at temperatures between about 50° C. and about 70° C. More preferably, high stringency conditions are 0.02 M sodium chloride, 0.5% casein, 0.02% SDS, 0.001 M sodium citrate, at a temperature of 50° C.
Example 22
Determination of Homologous and Degenerate Nucleic Acid Sequences
Modification and changes can be made in the sequence of the proteins of the present invention and the nucleic acid segments which encode them and still obtain a functional molecule that encodes a protein with desirable properties. The following is a discussion based upon changing the amino acid sequence of a protein to create an equivalent, or possibly an improved, second-generation molecule. The amino acid changes can be achieved by changing the codons of the nucleic acid sequence, according to the codons given in Table 10.







TABLE 10







Codon degeneracies of amino acids











One




Amino acid
letter
Three letter
Codons





Alanine
A
Ala
GCA GCC GCG GCT


Cysteine
C
Cys
TGC TGT


Aspartic acid
D
Asp
GAC GAT


Glutamic acid
E
Glu
GAA GAG


Phenylalanine
F
Phe
TTC TTT


Glycine
G
Gly
GGA GGC GGG GGT


Histidine
H
His
CAC CAT


Isoleucine
I
Ile
ATA ATC ATT


Lysine
K
Lys
AAA AAG


Leucine
L
Leu
TTA TTG CTA CTC CTG CTT


Methionine
M
Met
ATG


Asparagine
N
Asn
AAC AAT


Proline
P
Pro
CCA CCC CCG CCT


Glutamine
Q
Gln
CAA CAG


Arginine
R
Arg
AGA AGG CGA CGC CGG CGT


Serine
S
Ser
AGC AGT TCA TCC TCG TCT


Threonine
T
Thr
ACA ACC ACG ACT


Valine
V
Val
GTA GTC GTG GTT


Tryptophan
W
Trp
TGG


Tyrosine
Y
Tyr
TAC TAT









Certain amino acids can be substituted for other amino acids in a protein sequence without appreciable loss of enzymatic activity. It is thus contemplated that various changes can be made in the peptide sequences of the disclosed protein sequences, or their corresponding nucleic acid sequences without appreciable loss of the biological activity.
In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate/glutamine/aspartate/asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
It is known in the art that certain amino acids can be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are more preferred, and those within ±0.5 are most preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101 (Hopp, T. P., issued Nov. 19, 1985) states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0±1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine/histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4).
It is understood that an amino acid can be substituted by another amino acid having a similar hydrophilicity score and still result in a protein with similar biological activity, i.e., still obtain a biologically functional protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are more preferred, and those within ±0.5 are most preferred.
As outlined above, amino acid substitutions are therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Changes which are not expected to be advantageous may also be used if these resulted in functional patatin proteins.
Example 23
Production of Patatin Proteins and Permuteins in Plants
Plant Vectors
In plants, transformation vectors capable of introducing nucleic acid sequences encoding patatin proteins and permuteins are easily designed, and generally contain one or more nucleic acid coding sequences of interest under the transcriptional control of 5′ and 3′ regulatory sequences. Such vectors generally comprise, operatively linked in sequence in the 5′ to 3′ direction, a promoter sequence that directs the transcription of a downstream heterologous structural nucleic acid sequence in a plant; optionally, a 5′ non-translated leader sequence; a nucleic acid sequence that encodes a protein of interest; and a 3′ non-translated region that encodes a polyadenylation signal which functions in plant cells to cause the termination of transcription and the addition of polyadenylate nucleotides to the 3′ end of the mRNA encoding the protein. Plant transformation vectors also generally contain a selectable marker. Typical 5′-3′ regulatory sequences include a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. Vectors for plant transformation have been reviewed in Rodriguez et al. (Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston., 1988), Glick et al. (Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., 1993), and Croy (Plant Molecular Biology Labfax, Hames and Rickwood (Eds.), BIOS Scientific Publishers Limited, Oxford, UK., 1993).
Plant Promoters
Plant promoter sequences can be constitutive or inducible, environmentally- or developmentally-regulated, or cell- or tissue-specific. Often-used constitutive promoters include the CaMV 35S promoter (Odell, J. T. et al., Nature 313: 810-812, 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins et al., Nucleic Acids Res. 20: 8451-8466, 1987), the mannopine synthase (mas) promoter, the nopaline synthase (nos) promoter, and the octopine synthase (ocs) promoter. Useful inducible promoters include promoters induced by salicylic acid or polyacrylic acids (PR-1, Williams, S. W. et al, Biotechnology 10: 540-543, 1992), induced by application of safeners (substituted benzenesulfonamide herbicides, Hershey, H. P. and Stoner, T. D., Plant Mol. Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Lee et al., Proc. Natl. Acad. Sci. USA. 83: 6815-6819, 1986; Ainley et al., Plant Mol. Biol. 14: 949-967, 1990), a nitrate-inducible promoter derived from the spinach nitrite reductase gene (Back et al., Plant Mol. Biol. 17: 9-18, 1991), hormone-inducible promoters (Yamaguchi-Shinozaki, K. et al., Plant Mol. Biol. 15: 905-912, 1990; Kares et al., Plant Mol. Biol. 15: 225-236, 1990), and light-inducible promoters associated with the small subunit of RuBP carboxylase and LHCP gene families (Kuhlemeier et al., Plant Cell 1: 471, 1989; Feinbaum, R. L. et al., Mol. Gen. Genet. 226: 449-456, 1991; Weisshaar, B. et al., EMBO J. 10: 1777-1786, 1991; Lam, E. and Chua, N. H., J. Biol. Chem. 266: 17131-17135, 1990; Castresana, C. et al., EMBO J. 7: 1929-1936, 1988; Schulze-Lefert et al., EMBO J. 8: 651, 1989). Examples of useful tissue-specific, developmentally-regulated promoters include the β-conglycinin 7S promoter (Doyle, J. J. et al., J. Biol. Chem. 261: 9228-9238, 1986; Slighton and Beachy, Planta 172: 356-363, 1987), and seed-specific promoters (Knutzon, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. 89: 2624-2628, 1992; Bustos, M. M. et al., EMBO J. 10: 1469-1479, 1991; Lam and Chua, Science 248: 471, 1991; Stayton et al., Aust. J Plant. Physiol. 18: 507, 1991). Plant functional promoters useful for preferential expression in seed plastids include those from plant storage protein genes and from genes involved in fatty acid biosynthesis in oilseeds. Examples of such promoters include the 5′ regulatory regions from such genes as napin (Kridl et al., Seed Sci. Res. 1: 209-219, 1991), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACP desaturase, and oleosin. Seed-specific gene regulation is discussed in EP 0 255 378. Promoter hybrids can also be constructed to enhance transcriptional activity (Comai, L. and Moran, P. M., U.S. Pat. No. 5,106,739, issued Apr. 21, 1992), or to combine desired transcriptional activity and tissue specificity.
Plant Transformation and Regeneration
A variety of different methods can be employed to introduce such vectors into plant protoplasts, cells, callus tissue, leaf discs, meristems, etcetera, to generate transgenic plants, including Agrobacterium-mediated transformation, particle gun delivery, microinjection, electroporation, polyethylene glycol mediated protoplast transformation, liposome-mediated transformation, etcetera (reviewed in Potrykus, I. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225, 1991). In general, transgenic plants comprising cells containing and expressing DNAs encoding patatin proteins and permuteins can be produced by transforming plant cells with a DNA construct as described above via any of the foregoing methods; selecting plant cells that have been transformed on a selective medium; regenerating plant cells that have been transformed to produce differentiated plants; and selecting a transformed plant which expresses the protein-encoding nucleotide sequence.
Specific methods for transforming a wide variety of dicots and obtaining transgenic plants are well documented in the literature (Gasser and Fraley, Science 244: 1293-1299, 1989; Fisk and Dandekar, Scientia Horticulturae 55: 5-36, 1993; Christou, Agro Food Industry Hi Tech, p. 17, 1994; and the references cited therein).
Successful transformation and plant regeneration have been reported in the monocots as follows: asparagus (Asparagus officinalis; Bytebier et al., Proc. Natl. Acad. Sci. U.S.A. 84: 5345-5349, 1987); barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol. 104: 37-48, 1994); maize (Zea mays; Rhodes, C. A. et al., Science 240: 204-207, 1988; Gordon-Kamm et al., Plant Cell 2: 603-618, 1990; Fromm, M. E. et al., Bio/Technology 8: 833-839, 1990; Koziel et al., Bio/Technology 11: 194-200, 1993); oats (Avena sativa; Somers et al., Bio/Technology 10: 1589-1594, 1992); orchardgrass (Dactylis glomerata; Horn et al., Plant Cell Rep. 7: 469-472, 1988); rice (Oryza sativa, including indica and japonica varieties; Toriyama et al., Bio/Technology 6: 10, 1988; Zhang et al., Plant Cell Rep. 7: 379-384, 1988; Luo and Wu, Plant Mol. Biol. Rep. 6: 165-174, 1988; Zhang and Wu, Theor. Appl. Genet. 76: 835-840, 1988; Christou et al., Bio/Technology 9: 957-962, 1991); rye (Secale cereale; De la Pena et al., Nature 325: 274-276, 1987); sorghum (Sorghum bicolor; Casas, A. M. et al., Proc. Natl. Acad. Sci. U.S.A. 90: 11212-11216, 1993); sugar cane (Saccharum spp.; Bower and Birch, Plant J. 2: 409-416, 1992); tall fescue (Festuca arundinacea; Wang, Z. Y. et al., Bio/Technology 10: 691-696, 1992); turfgrass (Agrostis palustris; Zhong et al., Plant Cell Rep. 13: 1-6, 1993); wheat (Triticum aestivum; Vasil et al., Bio/Technology 10: 667-674, 1992; Weeks, T. et al., Plant Physiol. 102: 1077-1084, 1993; Becker et al., Plant J. 5: 299-307, 1994), and alfalfa (Masoud, S. A. et al., Transgen. Res. 5: 313, 1996); Brassica (canola/oilseed rape) (Fry, J. Plant Cell Rep. 6: 321-325, 1987); and soybean (Hinchee, M. Bio/Technol. 6: 915-922, 1988).
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations can be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.

## Claims

1. An isolated nucleic acid sequence encoding the protein of SEQ ID NO: 247.

2. The isolated nucleic acid sequence of claim 1, comprising SEQ ID NO: 246.

3. A recombinant vector operatively linked in the 5′ to 3′ orientation, comprising:
a) a promoter that directs transcription of a structural nucleic acid sequence; 
b) a nucleic acid sequence encoding the protein of SEQ ID NO: 247; and 
c) a 3′ transcription terminator.

4. The recombinant vector of claim 3, wherein the vector expresses a protein exhibiting corn rootworm insect inhibitory activity and acyl lipid hydrolase activity.

5. A recombinant host cell comprising a nucleic acid sequence encoding the protein of SEQ ID NO: 247.

6. The recombinant host cell of claim 5, wherein the protein of SEQ ID NO: 247 exhibits corn rootworm insect inhibitory activity and acyl lipid hydrolase activity.

7. A recombinant plant comprising a nucleic acid sequence encoding the protein of SEQ ID NO: 247.

8. The recombinant plant of claim 7, wherein the protein of SEQ ID NO: 247 exhibits corn rootworm insect inhibitory activity and acyl lipid hydrolase activity.