Characterization of Pen n 13, a major allergen from the mold Penicillium notatum

Penicillium notatum is a well-known indoor aeroallergen and is frequently included in skin test panels for allergic diagnosis. On two-dimensional immunoblotting using patients' sera containing IgE and monoclonal antibody D7B8 specific for Pen c 1 of P. citrinum, two allergens with a molecular mass of 33 kDa but different isoelectric points were identified. A novel cDNA coding for Pen n 13 was cloned and sequenced. The nucleotide sequence codes for a protein 397 amino acids including a putative signal peptide of 25 amino acids and a propeptide of 90 amino acids. The allergen is an alkaline serine protease that shares more than 39% identical residues with other kinds of mold allergens. The coding cDNA of Pen n 13 was cloned into vector pQE-30 and expressed in E. coli M15 as a His-tag fusion protein and purified to homogeneity. The fusion protein reacted with monoclonal antibodies of Pen c 1 and with IgE from Penicillium-allergic patients. Furthermore, it also cross-reacted strongly with IgE specific for the natural Pen c 1, indicating that similar IgE binding epitopes may exist in the allergens of P. notatum and P. citrinum. Antigenicity index plots indicated that there are several similar epitope regions of high antigenic indices in Pen c 1 and Pen n 13, corroborating that mold allergens belonging to the alkaline serine protease family possess similar protein structure and strong antigenic cross-reactivity.     

Reduced allergenic potency of VR9-1, a mutant of the major shrimp allergen Pen a 1 (tropomyosin)

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90-98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.     

Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology.

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L. destructor-sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and express new allergens from L. destructor and determine their recognition frequency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor-sensitized individuals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His6-tagged proteins. Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L. destructor-sensitized subjects, respectively.     

Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Snow crab (Chionoecetes opilio) proteins have been recognized as an important source of both food and occupational allergens. While snow crab causes a significant occupational allergy, only one novel allergen has recently been fully characterized. The muscle proteins from snow crab legs were profiled by SDS-PAGE. Several of these proteins were characterized using tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20kDa), arginine kinase (40), troponin (23kDa) and α-actine (42kDa) and smooth endoplasmic reticulum Ca(2+)ATPase (113kDa). Immunoblotting using serum of sixteen allergic patients resulted in strong reactivity with the 40-kDa protein in seven patients (43%). This protein was purified by chromatography and subsequently de novo sequenced using matrix assisted laser desorption ionization and electrospray tandem mass spectrometry. We identified a second important allergen, arginine kinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for arginine kinase. The suitability of this signature peptide was tested for analytically representing the arginine kinase, by performing a multi-reaction monitoring tandem mass spectrometry approach on actual air filter samples collected from a simulated crab processing plant.     

Molecular cloning and expression of Cro s 1: an occupational allergen from saffron pollen (Crocus sativus)

Background:

The cultivation of saffron is expanding through the southeast of Iran, and allergy to saffron pollen occurs in workers involved in processing this plant. We aimed to clone, sequence and express a major allergen involved in saffron pollen allergy, and to compare the recombinant with the natural allergen.

Methods:

The N-terminal amino acid sequence of Cro s 1, an allergen from saffron pollen, was determined after immunoblotting. The cDNA encoding for this allergen was cloned by PCR utilizing a primer based on the N-terminal amino acid sequence. Recombinant Cro s 1 (rCro s 1) was expressed as a soluble protein in Pichia pastoris and purified to homogeneity by gel filtration. Inhibition of IgE binding to rCro s 1 by pollen extract was analyzed by ELISA.

Section Title

The allergen Cro s 1 was identified from saffron pollen extracts and cloned by PCR. Cro s 1 cDNA defined an acidic polypeptide with homology to pollen proteins from Chenopodium album and Ligastrum vulgaris. The rCro s 1 was expressed in P. pastoris at 28 mg/l. Saffron pollen extract inhibited the binding of patient serum IgE to rCro s 1.

Conclusion:

We identified and cloned the first Crocus sativus pollen allergen. rCro s 1 cDNA shows a very high homology with Che a 1, the major allergen of lamb''s-quarter, Chenopodium album, Caryophyllales, pollen (97%). Cro s 1 is a useful tool for specific diagnosis and structural studies of occupational allergy to saffron.Key Words: Allergen, cDNA cloning, Cro s 1, Occupational allergy, Saffron pollen     

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.     

Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen.

IgE recognition of indoor allergens represents a major cause of allergic asthma in atopic individuals. We found that 52 of 102 patients suffering from allergic symptoms indoors contained IgE Abs against allergens from the Indianmeal moth (Plodia interpunctella), a ubiquitous food pest. Using serum IgE from a moth-sensitized patient we screened an expression cDNA library constructed from P. interpunctella larvae. cDNAs coding for arginine kinase (EC 2.7.3.3), a 40-kDa enzyme commonly occurring in invertebrates that is involved in the storage of such high-energy phosphate bonds as phosphoarginine, were isolated. Recombinant moth arginine kinase, designated Plo i 1, was expressed in Escherichia coli as a histidine-tagged protein with enzymatic activity, and purified to homogeneity by nickel chelate affinity chromatography. Purified recombinant arginine kinase induced specific basophil histamine release and immediate as well as late-phase skin reactions. It reacted with serum IgE from 13 of the 52 (25%) moth-allergic patients and inhibited the binding of allergic patients' IgE to an immunologically related 40-kDa allergen present in house dust mite, cockroach, king prawn, lobster, and mussel. Our results indicate that arginine kinases represent a new class of cross-reactive invertebrate pan-allergens. Recombinant arginine kinase may be used to identify a group of polysensitized indoor allergic patients and for immunotherapy of these individuals.     

Cloning and expression of cyclophilin from Platanus orientalis pollens in Escherichia coli

Background:

Allergy is a clinical disorder affecting the human population with wide geographical distribution. Platanus orientalis (P. orientalis) trees are planted in many countries and their pollen causes allergic reactions.Cyclophilin has recently been identified as one of the most important allergens of P. orientalis pollen. We aimed to clone and purify this allergen in Escherichia coli for further studies and therapeutic and diagnostic purposes for allergy to P. orientalis.

Methods:

RNA was extracted from P. orientalis. A full-length fragment encoding cyclophilin was prepared by polymerase chain reaction amplification of the first-strand cDNA synthesized from P. orientalis RNA. The cDNA was inserted into the pET32b (+) vector, and the construct transformed into E. coli Top10 and BL21 cells. The expressed protein was purified by the CuSO4 method.

Results:

The cDNA for the cyclophilin of P. orientalis pollen was cloned, and a specific reactivity of recombinant cyclophin was confirmed by immunoblotting using sera from patients allergic to P. orientalis pollen.

Conclusion:

The recombinant cyclophilin has a potential for immunologic assays for evaluation of allergy to P. orientalis pollen.Key Words: Allergy; Recombinant allergen; Cyclophilin, Escherichia coli, Platanus orientalis, Pollen, Cloning     

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.     

B cell epitopes of the major timothy grass pollen allergen, phl p 1, revealed by gene fragmentation as candidates for immunotherapy.

Group 1 grass pollen allergens are recognized by IgE antibodies of almost 40% of allergic individuals and therefore belong to the most important elicitors of Type I allergy worldwide. We have previously isolated the cDNA coding for the group 1 allergen from timothy grass, Phl p 1, and demonstrated that recombinant Phl p 1 contains most of the B cell as well as T cell epitopes of group 1 allergens from a variety of grass and corn species. Here we determine continuous B cell epitopes of Phl p 1 by gene fragmentation. IgE antibodies of grass pollen allergic patients identified five continuous epitope-containing areas that on an average bound 40% of Phl p 1-specific IgE antibodies and were stably recognized in the course of disease. In contrast to untreated patients, patients undergoing grass pollen immunotherapy started to mount IgG(4) antibodies to the recombinant IgE-defined fragments in the course of immunotherapy. The protective role of these IgG(4) antibodies is demonstrated by observations that 1) increases in rPhl p 1 fragment-specific IgG(4) were in parallel with decreases in Phl p 1-specific IgE, and 2) preincubation of rPhl p 1 with patients sera containing rPhl p 1 fragment-specific IgG(4) blocked histamine release from basophils of an untreated grass pollen allergic patient. We propose to use recombinant Phl p 1 fragments for active immunotherapy in order to induce protective IgG responses against IgE epitopes in grass pollen allergic patients. This concept may be applied for the development of allergy vaccines whenever the primary sequence or structure of an allergen is available.     

Thioredoxin from the Indianmeal Moth Plodia interpunctella: Cloning and Test of the Allergenic Potential in Mice

Background/Objective

The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified.

Objective

The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1.

Method

A cDNA library from whole adult P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients'' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured.

Result

For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation.

Conclusion

Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new P. interpunctella allergen.     

Gastrointestinal digestion and absorption of Pen j 1, a major allergen from Kuruma prawn, Penaeus japonicus

Tropomyosin had been identified as a major allergen in shrimp. The digestion and absorption of tropomyosin (Pen j 1) from kuruma prawn were investigated by ex vivo, in vitro, and in vivo techniques in order to elucidate the relationship between the allergenicity of the allergen and its gastrointestinal behavior. Pen j 1 transported the Caco-2 monolayer in a dose-dependent manner, and also enhanced the permeability of lucifer yellow, a marker of paracellular transportation, at high concentrations of the allergen. Studies with everted sacs revealed that Pen j 1 was rapidly degraded to small peptides (MW<3.5 kDa) and amino acids by intestinal proteases and absorbed from enterocytes. Furthermore, Pen j 1 orally administered to rats tended to remain in the stomach rather than in the small intestine, after which the allergen moved to the epithelial cells. These observations suggest that Pen j 1 may be absorbed via the gastric mucosa prior to its digestion in the intestines.     

Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3

BackgroundDust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust.ObjectiveOur aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis.MethodsA Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers.ResultsIn addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test.ConclusionsIn addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy.     

Molecular and immunological characterization of tri a 36, a low molecular weight glutenin, as a novel major wheat food allergen

Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ～80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.     

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.     

Identification of a villin-related tobacco protein as a novel cross-reactive plant allergen

In a paradigmatic approach we identified cross-reactive plant allergens for allergy diagnosis and treatment by screening of a tobacco leaf complementary DNA (cDNA) library with serum IgE from a polysensitized allergic patient. Two IgE-reactive cDNA clones were isolated which code for proteins with significant sequence similarity to the actin-binding protein, villin. Northern- and Western-blotting demonstrate expression of the villin-related allergens in pollen and somatic plant tissues. In addition, villin-related proteins were detected in several plant allergen sources (tree-, grass-, weed pollen, fruits, vegetables, nuts). A recombinant C-terminal fragment of the villin-related protein was expressed in Escherichia coli, purified and shown to react specifically with allergic patients IgE. After profilin, villin-related proteins represent another family of cytoskeletal proteins, which has been identified as cross-reactive plant allergens. They may be used for the diagnosis and treatment of patients suffering from multivalent plant allergies.     

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.     

Elevation of soluble interleukin-2 receptor levels in nasal allergy

To investigate soluble IL-2 receptor (sIL-2R) levels in nasal allergy, the sera and nasal secretions from patients with nasal allergy and from healthy subjects were subjected to a double-epitope enzyme-linked immunosorbent assay. Significant elevation of sIL-2R concentrations in the sera and nasal secretions was observed in the allergy patients (n = 26) compared with those of healthy subjects (n = 9). IL-2R-positive (CD25(+)) cells were observed in the crust formed in an allergic nasal mucosa. The concentration of sIL-2R in the sera correlated neither with the eosinophil count of the peripheral blood count nor with clinical severity. The concentration of sIL-2R in the nasal secretions was significantly higher compared with that in the sera from allergic patients (p < 0.01), whereas no significant difference was observed between sIL-2R levels in the sera and nasal sections from normal subjects. These findings indicate that sIL-2R plays an essential role in allergic processes by regulating IL-2R-positive cells recruited into the nasal mucosa.     

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family

Pear is known as an allergenic food involved in the ‘oral allergy syndrome’ which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n=16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.