Disclosed is a method for identification of key amino acids in plant proteins critical in generating allergenic activity through mapping the epitope(s) harboring human IgE binding activity. The identified epitope(s) are then modified by amino acid substitution preferably by alanine substitution, for reduced or negative IgE-binding activity. A plant gene expression system comprising a DNA construct placed operably under the control of a promoter sequence that confers seed-specific expression is also disclosed for the expression of the modified proteins. The Brazil nut 2S sulfur-rich protein was exemplified. The method disclosed herein is particularly useful for the production of dietary proteins with improved nutritional quality and reduced or negative allergenicity for human and animal consumption through genetic engineering.

Description of the Invention

SUMMARY OF THE INVENTION

In the invention, the target proteins can be of diverse origins. They may possess biological or pharmaceutical functions and can be applied for human and livestock consumption. Taking advantage of an exceptionally high content (18%) of methionine and an allergenic nature, the Brazil nut methionine-rich protein (BNMRP) was adopted as an example target protein for the production of a sulfur-rich protein with a reduced or negative allergenic activity.

The invention is to provide a systematic method for obtaining a fine IgE epitope mapping of a target protein using the inventive merged strategy of recombinant, overlapping peptides to thereby identify amino acids important for IgE binding within the epitope.

Accordingly, a first aspect of the invention is to provide a modified methionine- and/or cysteine-rich protein having at least one epitope binding to human IgE comprising arginine, in which in the modified protein, the arginine is substituted with alanine or a residue thereof, or with methionine or a residue thereof, so that the modified protein has a reduced or negative allergenic activity.

A second aspect of the invention is to provide a nucleic acid sequence encoding a modified protein defined herein.

According to a third aspect of the invention, there is provided a transgenic plant and/or progeny thereof comprising a modified protein defined herein.

According to a fourth aspect of the invention, there is provided a method for reducing or eliminating human IgE-binding activity of a protein, the protein having at least one epitope binding to human IgE comprising arginine or alanine, the method comprising: identifying the epitope of the protein; and replacing arginine in the epitope with alanine or a residue thereof, or replacing alanine with methionine or a residue thereof.

According to a sixth aspect of the invention, there is provided a host cell comprising a DNA construct defined herein.

According to a seventh aspect of the invention, there is provided a method for constructing a transgenic plant comprising: a) constructing a DNA construct comprising a nucleic acid defined herein operably linked to a vector; b) transfecting a plant cell with the construct; and c) producing the transgenic plant from the plant cell.

An eighth aspect of the invention is directed to use of a modified protein defined herein in preparing food for animal consumption.

The IgE-binding activity, i.e. the allergenic activity of the modified protein according to the invention and a plant protein produced by the method of the invention can be significantly reduced or eliminated. Therefore, the target protein of the invention can be applied for human and livestock consumption.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a modified protein (target protein) and a plant gene expression system which comprises a DNA construct placed operably under the control of a promoter sequence that confers a seed-specific expression. The DNA construct contemplated herein encodes one or more modified proteins and their derivatives which can significantly reduce or negative human IgE-binding activity.

The target protein that can be used in the invention includes those methionine- and/or cysteine-rich proteins, such as a protein of the 2S family of albumin proteins in Brazil nut. Such a modified protein may be used for safer human and livestock consumption and for immunotherapy of brazil-nut allergy by reducing its anaphylactic side effects.

The 2S albumins are storage proteins present in diverse species. Despite high variability in amino acid sequences, the 2S albumins share a similar structure that is heterodimeric and consisting of a large subunit and a small subunit synthesized as a single precursor polypeptide. All the 2S albumins are compact globular proteins with conserved cysteine residues, which are responsible for the disulfur bonds linking the small and the large polypeptides and forming the whole protein. The 2S proteins are abundant in seeds of the plant, providing reserved amino acids during the seed germination and seedling growth.

However, many 2S albumins have been identified as major allergens. The potential to introduce new allergenic proteins into food through plant genetic engineering is of a great concern to public. This issue was first highlighted by our previous study with a 2S methionine-rich protein from Brazil nut (BNMRP). Many other proteins with biological activities, which could have biotechnological applications to improve food quality or to confer improved agronomic performance or resistance to the plant, are also known allergens.

Therefore, carefulness must be taken in choosing safe proteins to use in plant biotechnology, and systems to assess the allergenic potential of foods derived from genetically engineered crop plants have to be in place and enforced to ensure that the potential allergenic proteins are identified before entering the food chain. An attractive alternative is to modify or to design and generate non-allergenic proteins with similar structures and biological properties by genetic engineering for plant improvement.

In this invention, the inventors have adopted the BNMRP as an example for the elucidation and characterization of the IgE epitopes on the target protein and hence, provide significant understanding of the allergenic nature of the 2S allergens. The inventors have successfully demonstrated that it is feasible to modify a protein to be one having a reduced allergenic activity or no longer triggering allergy reactions. This opens up a new approach to enhance the quality of legume proteins without allergenicity, through transferring and expressing the modified methionine-rich and/or cystine-rich protein gene(s) for animal feed or human consumption, and restores the public confidence in genetically modified products.

The overall strategy of this invention includes: 1) mapping an epitope of the target protein; 2) identifying amino acids critical to a human IgE binding within the epitope; 3) modifying the epitope and/or related domains to reduce or negative the IgE binding; 4) establishing a plant gene expression system with a recombinant protein from the target protein for the accumulation of the target protein with reduced allergenicity; and 5) confirming the expressed recombinant protein having a reduced or negative allergenic activity.

It is well known that the interaction between an allergen and immune system at the molecular level plays a crucial role in the etiology of allergy. The present invention provides a systematic strategy to identify IgE-binding epitopes on an allergen so that the nature of allergenicity of the allergen can be elucidated.

The inventors have developed a new strategy to identify epitopes on a target proteins by recombinant, overlapping peptides, which merges the advantages of the two existing methods described in the prior art. The recombinant, overlapping peptides, like synthetic, overlapping peptides, ensure a systematic coverage of the entire allergen sequence, whereas it is not possible to ensure the entire allergen sequence is represented in the peptide library. The peptide length of the recombinant, overlapping peptides in the current strategy is not limited to 15 residues as in the case of the SPOTs system, so that it may allow the identification of at least some conformational epitopes. It is also easier to produce and purify the recombinant peptides in large amounts for multiple immunodetections simply by growing and inducing more E. coli cells containing the recombinant peptide constructs. Since the recombinant peptides may be fused to trxA at the C-terminus, it is efficient to generate point mutations on the recombinant IgE binding epitopes by PCR, for side-by-side comparison between an unmodified epitope and a mutated epitope. Through this systematic approach, the inventors have identified at least 8 IgE epitopes on one of target proteins, BNMRP, for example, which subsequently allowed the reduction and removal of the IgE binding ability of the methionine-rich allergen by site-directed mutagenesis (described below).

Construction of Recombinant Gene Fragments of Target Proteins

Gene-specific primers for amplifying the target fragments are first designed with restriction enzyme sites for cloning into an expression vector.

Various combinations of primers are applied for amplifying the fragment encoding different gene fragments of the target protein. After sequenced, the PCR products are cloned in an expression vector through the restrictive enzyme sites designed in the primers such as cloned into a pET-30a(+) vector (Novagen) to create different constructs which can express different fusion proteins such as His.tag::BNMRPs::trxA::fliC. A protein without a fragment for target protein is also amplified as a control.

Construction of N-Terminal Deletions of Target Proteins

The clones containing the above cloned fragments are used as templates for producing deletions of the target protein. To generate N-terminal deletions, a series of overlapping primers, offset nine nucleotides encoding 3 amino acids of the target protein are designed. Deletions with a progressive 3-amino acid truncation are generated by PCR with template plasmids. The PCR products are subsequently cloned into a expression vector to form deletion fusion proteins such as His.tag::deletions::trxA::fliC.

Construction of Overlapping, Recombinant Peptides

For generating overlapping, recombinant peptides, each 3' primer is designed with a stop codon to truncate the 3' region of the deletion. The recombinant peptides are constructed by PCR using the combinations of the 3' primers and a promoter primer in the vector (Promega). The amplified fragments are then subcloned into an expression vector, producing small overlapping peptides fused to the C-terminal of a tag.

The recombinant constructs can be expressed in a host cell such as E. col. and the recombinant proteins can be purified according to the tags in the fusion proteins. Alternatively, the recombinant proteins can be detected using the method of SDS-PAGE with Coomassie brilliant blue staining or immunoblotting as an example.

Epitope Mapping of Target Proteins

Recombinant proteins containing N-terminal serially deleted proteins, or overlapping peptides of a target protein can be purified and immuno-detected with an anti-polyclonal antibody, or human serum from patients allergic to the target protein. At least 8 linear epitopes have been identified in the BNMRP, one of the preferred embodiments of the invention. An epitope with the strongest IgE reactivity can be consequently mapped.

To define the precise position and sequences of epitopes recognized by IgE human serum, the inventors further generates a series of overlapping recombinant peptides covering the entire length of the target protein where IgE binding is observed previously by the deletion approach. In one preferred embodiment, each peptide is 7-8 amino acids in length and progressively offsetting from the previous peptide by 3 amino acids. This approach allows a systematic analysis of the primary sequence of the entire target protein to determine the exact amino acid sequences of the IgE binding regions. For example, as shown in FIG. 3 (see Original Patent), eight epitopes, designated S0 and S1 on a small subunit and L1-L6 on a large subunit of BNMRP, have been identified in this manner.

Identification of Key Amino Acids Critical to IgE Binding within Identified Epitopes

Amino acid substitution analysis of the epitopes shows that mutation of key amino acids to alanine could significantly reduce or eliminate IgE binding. A series of mutants are generated for the identified epitopes by oligonucleotide-mediated mutagenesis using PCR. In each mutant, a selected single amino acid in the native epitope is substituted with an alanine or a residue thereof. If the native amino acid is an alanine, it is replaced by a methionine. This approach allows elucidation of amino acids required in ligand binding within each epitope, since systematic substitution of amino acids with alanine eliminates side chains binding to antibody.

Using the clones containing the native epitopes as templates, PCR is carried out by combination of the vector promoter primer such as a T7 primer and a primer designed to introduce the mutation. Then the PCR products are ligated with a backbone of expression vectors, forming a fusion protein containing the mutated epitope.

The recombinant peptides are probed with an allergic patient's serum to determine the effect of a single amino acid change on the target protein-specific IgE binding. When alanine is substituted for a wild-type amino acid at the position of Arg, the mutated peptide is not recognized by the human serum, or a decrease in binding is observed in the embodiment of the invention. Therefore the mutant position could be identified as a key amino acid critical to IgE binding within the identified epitope.

In the work leading to the present invention, the inventors have demonstrated that arginine or a residue thereof is crucial for IgE binding with the epitope on the target protein such as BNMRP, since all identified epitopes on this molecule contain arginine residues and point mutations of this amino acid within each IgE epitope to alanine result in, without exception, a dramatic decrease or a loss in antibody binding. This positively charged amino acid is the second abundant amino acid (14.85%) in the BNMRP and spreads over the whole protein molecule. The change of the arginine residues by mutation may alter the surface charge as well as the conformational structure of the epitopes or the whole protein molecule, consequently leading to a reduction or a loss in its IgE binding with IgE. This is the first report that a conserved amino acid (Arg) in the epitopes of a food allergen involves in IgE binding.

It is also worth to note that a common structure Arg-Cys, and amino acid sequence Met-Arg harbor IgE binding ability. The sequence similarities suggest the presence of a cross-reacting IgEs capable of recognizing both epitopes on the same protein. This helps to explain the potent nature of the methionine-rich allergen, as in the case of Hev b 5 where in the IgE epitopes 5.7 and 5.8, both having the sequence EKPAE (SEQ ID NO: 178), are cross-reactive (Beezhold D. H. et al., 2001). Although common structural characteristics of linear IgE epitopes are limited so far, the situation may change when more epitope mapping results come in.

Modifications of Epitopes and Related Domains for Reduced Target Protein Specific IgE Binding

The present invention encompasses a systematic method for the generation of a derivative of the target protein with a greatly reduced IgE reactivity by point mutation of the identified linear epitopes.

Proteins that can be used in the invention include those rich in methionine and/or cysteine, such as the 2S family of proteins including a protein from Brazil nut, amongst others. Such a modified protein may be used for a safer human and livestock consumption and for immunotherapy.

Through the application of this systematic approach, the present invention demonstrates that the IgE binding ability of the epitopes can be reduced or removed by mutation of the arginine rather than the methionine in the epitopes, providing the possibility that a modified target protein with reduced IgE binding ability can be generated by mutations without decreasing its methionine content.

Generation of Gene Constructs Encoding Foreign Target Protein with Reduced Allergenicity for Plant Expression

The present invention further encompasses a plant gene expression system comprising a DNA construct placed operably under the control of a promoter sequence that confer seed-specific expression. The DNA construct contemplated herein encodes one or more subunits of a sulfur rich-2S seeds storage protein with significantly reduced IgE-binding activity through modifications, more preferably, alanine substitution of the identified epitopes.

The identified epitopes of proteins are modified by site-directed mutagenesis using PCR to generate a specific point mutation for alanine. The PCR products containing the mutations are linked together through the restrictive enzyme site to generate constructs containing a nucleic acid sequence encoding the mutations in epitopes using a similar strategy as described above. Alternatively, some successive overlap extension PCR reactions can be carried out to introduce further specific point mutations in the epitopes. The constructs containing a nucleic acid sequence encoding at least one mutation are introduced into competent host cells for further plant application.

In one embodiment, through the application of the inventive strategy for oligonucleotide-directed mutagenesis, the inventors have generated a recombinant BNMRP clone with mutated epitopes as an example. The inventors chose amino acids in the epitopes that, when changed, resulted in the greatest reduction in IgE binding. Most of the selected amino acids are mutated to alanine. However, in an illustrating engineering, some arginines encoded by the codon AGG in the cDNA are mutated to methionine. Thus, the methionine content of the modified BNMRP is simultaneously increased after mutation.

The relative extent of IgE binding to the altered sequence can be analyzed by SDS-PAGE and probe hybridization with allergic patients' serum against target protein and assessed by densitometry scanning and compared with that of the native one.

The present invention also extends to further engineered variants, including modified proteins with cysteine residues restored, and a lysine-rich protein (e.g. WBLRP) fusion, that are constructed and transformed into target plants for expression such as tobacco for seed-specific expressions.

Different types of plant species, including monocots and dicots, and various transformation techniques can be adopted for the present invention. However, it is preferred to use a plant that can be transformed with high transformation efficiency. Expression vectors containing the target protein expression cassettes can be introduced into plants according to known techniques such as Agrobacterium-mediated plant transformation, vacuum infiltration, gene transfer into pollen or calli or protoplast transformation (Bechtold N., et. al., 1993; Fisher D. K. and Guiltinan M. J., 1995). An ordinary skilled person in the art can make use of different strains of bacteria and transformation methods for the transformation of different host plants according to known techniques.

Plant regeneration is well known in the art. Transformants screened for desirable gene products are used for regeneration. The regenerated shoots (leaf-disc technique) or green plants (vacuum infiltration) are transferred in soil and grown in a green house for further expression analysis.

One of the objectives involves the application of a plant seed-specific phaseolin promoter and terminator region to the transgenes, which confines the transgenic expression only in the plant seeds. Another characteristic of this method involves the inclusion of an NPT II and a GUS gene, both driven by a 35S promoter and an NOS terminator. These two genes enable selection of positive transformants during the regeneration of new transgenic plants from calli, and further screening of possible transformants after the regeneration of plant leaves. In one preferred embodiment, all the components are put together into a pBI121 vector, which is an Agrobacterium tumefaciens-Ti plasmid system. The inventors has successfully provided in an example a method to make constructs for the transgenic plant seed-specific expression of different variations of MBNMRP. The modified proteins that can be expressed in transgenic plant seeds using this method include the various modified target proteins.

In one example, the inventors have used tobacco (Nicotiana tabacum) as the transformation host, since it is well established as a plant model system, and can be easily transformed via Agrobacterium-mediated method.

To investigate whether the transgenes integration and expression of recombinant proteins with reduced negative allergenic activity in plants are present in the regenerated tobacco plants, genomic PCR screening, Northern blot analysis for the RNA expressed in the transgenic plants, Western blot analysis for the proteins produced by the engineered variants for example, are performed in the invention. As a result, a plant expressing foreign target protein with reduced allergenicity has been confirmed.

To test if the allergenic activities of these transgenic proteins are hampered, a simulated gastric digestion method is introduced as an example. The inventors have found that MBNMRP in one example showed a significant decrease in thermo-stability than BNMRP, which may reflect a decrease allergenic potential produced by the method of the invention.
 

Claim 1 of 1 Claim

1. A method for reducing or eliminating the human IgE-binding activity of a protein comprising SEQ ID NO: 175 having at least one epitope binding to human IgE, comprising modifying the amino acids in the protein to produce a modified protein having the sequence of SEQ ID NO:177, wherein the modified protein exhibits reduced or eliminated IgE binding activity.

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