Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.     

Expression and characterization of three important panallergens from hazelnut

Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.     

牛奶过敏原制备方法的研究和免疫活性鉴别

目的探索牛奶主要过敏原的制备工艺。方法采用等电点沉淀、凝胶层析和分子筛等技术纯化牛奶中主要过敏原组分;采用SDS-PAGE鉴定蛋白纯度,采用双抗原夹心-ELISA鉴定免疫活性。结果成功获得牛奶中的四种主要过敏原组分:酪蛋白、β乳球蛋白、α乳白蛋白和分子量较高的P1组分,并经过免疫实验证实这四种组分均能与过敏血清产生反应,而其中β乳球蛋白和P1组分反应性较高。结论本研究探索出一种简单、实用的牛奶主要过敏原制备工艺,并证实牛奶中的β乳球蛋白和高分子量蛋白质为主要过敏原。     

Identification of hemocyanin as a novel non-cross-reactive allergen from the giant freshwater shrimp Macrobrachium rosenbergii

Scope : Sensitization to giant freshwater shrimp Macrobrachium rosenbergii (Mr) was recently reported. However, the allergens have yet to be identified. This study aimed to identify and characterize a novel allergen of Mr shrimp. Methods and results: Extracted proteins were separated and purified by anion and in some experiments, size‐exclusion chromatography. Serum IgE from shrimp allergic donors identified a candidate protein, which was characterized by LC‐MS/MS. The specificity of IgE binding was tested using immunoblotting and inhibition ELISA. The IgE‐binding profiles from 12 of 13 Mr allergic subjects that were pre‐incubated with an extract of Penaeus monodon showed residual binding to ～60–80 kDa proteins. The 60–80 kDa IgE‐bound proteins were fractionated in the flow‐through of anion chromatography showing a high IgE reactivity. Peptides identified by LC‐MS/MS showed the proteins closely match subunits of hemocyanin (Hcs). Purified Hcs from hemolymph markedly inhibited binding of IgE from sera of Mr allergic subjects to solid‐phased Mr proteins in inhibition ELISA. Conclusion: Hcs were identified as heat‐stable, non‐cross‐reactive, high‐molecular‐weight (MW) allergens from Mr shrimp. Since circulatory organs are not always removed during food preparation, high concentrations of Hcs may be present along with shrimp meat, which contains the known cross‐reactive tropomyosin protein.     

The panel of egg allergens, Gal d 1-Gal d 5: Their improved purification and characterization

Egg proteins represent one of the most important sources evoking food allergic reactions. In order to improve allergy diagnosis, purified and well-characterized proteins are needed. Although the egg white allergens Gal d 1, 2, 3 and 4 (ovomucoid, ovalbumin, ovotransferrin, and lysozyme) are commercially available, these preparations contain impurities, which affect exact in vitro diagnosis. The aim of the present study was to set up further purification protocols and to extend the characterization of the physicochemical and immunological properties of the final batches. The egg white allergens Gal d 1-4 were purified from commercial preparations, whereas Gal d 5 (alpha-livetin) was purified from egg yolk. The final batches of Gal d 1-5 consisted of a range of isoforms with defined tertiary structure. In addition, the IgE binding capacity of the purified egg allergens was tested using allergic patients' sera. The allergen batches will be further used to set up allergen specific diagnostic assays and to screen a larger collection of patients' sera.     

Quantitative analysis of shrimp allergen in food matrices using a protein chip based on sandwich immunoassay

Food allergy is increasingly becoming a serious concern these days. With packaged foods becoming the norm of the day, food allergy cases out of accidental consumption are becoming rampant, thereby generating great risks for the subjects involved and prompting food authorities in different countries to formulate new regulations about displaying food allergen data on food labels. Detection of food allergens is conventionally carried out by ELISA or PCR tests. These techniques are limited in that they can only detect one or few allergens at one time. Therefore, in the present study a novel sandwich protein chip assay was developed for quantitation of shrimp allergens in food matrixes. The shrimp allergen model used 3D aldehyde slides as the solid carrier, rabbit antisera as the capture reagent, and biotin-labeled monoclonal antibody as the detector reagent. Resulting antigen–antibody complexes were visualized in the presence of commercial strepavidin labeled with Cy3 to produce fluorescence for quantification. With the LOD of the protein chip being 0.054 mg tropomyosin/kg, the protein chip can quantify down to 0.096 mg tropomyosin/kg. The protein chip was not found to be sensitive to other kinds of foods but cross-reacted to some extent with allergens of some other crustaceans. The recoveries ranged from 69.2 to 99.9%, while the intra- and inter-assay coefficients of variation were <13% and <19%, respectively. It seems that the new assay is reliable enough to detect shrimp allergens in food and food products and help minimize the instances of shrimp allergy. It is also possible to use the protein chip for simultaneous detection of other food allergens.     

Production and characterization of an allergen panel for component-resolved diagnosis of celery allergy

In celery a relevant food allergen source, three allergens have been identified so far: Api g 1 and Api g 4, and one glycosylated protein, Api g 5. For component-resolved food allergy diagnosis high amounts of well-defined allergens are needed. Depending on the individual celery allergen, protocols for heterologous production and purification from natural source, respectively, were established to obtain homogenous protein batches. Afterwards the purified recombinant allergens, Api g 1, Api g 4 and natural Api g 5 were characterized regarding their structural integrity and immunological activity. Therefore, several methods were applied. Proteins were identified by partial N-terminal sequencing, protein mass was verified by MS and sequence integrity by MALDI-TOF and N-terminal sequencing after tryptic digestion. Presence of isoforms in natural allergen preparations was identified by 2-DE. Secondary and tertiary structures were evaluated by circular dichroism (CD) spectroscopy and NMR analysis. Finally, IgE binding capacity was verified using selected sera from celery allergic patients in IgE immunoblots and IgE ELISA. These well-defined celery allergens will be used to prove the concept of component-resolved diagnosis and will contribute to improve food allergy diagnosis in the future.     

Recombinant tropomyosin from Penaeus aztecus (rPen a 1) for measurement of specific immunoglobulin E antibodies relevant in food allergy to crustaceans and other invertebrates

Immunoglobulin E (IgE)-mediated food allergy to crustaceans and mollusks is relatively common and affected individuals typically react to a range of different species. The only known major allergen of shrimp was first described over 20 years ago and later identified as the muscle protein tropomyosin. This protein may be useful as a defined and relevant diagnostic marker for allergic sensitization to invertebrate foods. In order to generate an assay reagent suitable for this purpose, tropomyosin from the shrimp Penaeus aztecus (Pen a 1) was produced as a recombinant protein in Escherichia coli and characterized with respect to IgE antibody binding properties in comparison to natural shrimp tropomyosin. Hexahistidine-tagged rPen a 1 accumulated as a predominantly soluble protein in the E. coli expression host and a two-step chromatographic procedure provided a high yield of pure and homogeneous protein. rPen a 1 displayed chromatographic and folding characteristics similar to those of purified natural shrimp tropomyosin. Serum preincubation with serial protein dilutions revealed similar capacity of recombinant and natural tropomyosin to compete with immobilized shrimp extract for IgE binding. rPen a 1 was further shown to extensively and specifically compete for IgE binding to extracts of other crustacean species, house dust mite and German cockroach.     

融合型原肌球蛋白MBP-CTB-TM构建及其口服致敏性评价

将麦芽糖结合蛋白（maltose-binding protein,MBP）、霍乱毒素B亚基（cholera toxin B subunit,CTB）以及增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）基因重组构建MBP-CTB-EGFP并原核表达纯化,利用HEK293T细胞模型探究MBP-CTB-EGFP穿透细胞膜的能力,从而开发新型黏膜佐剂。进一步地,利用原肌球蛋白（tropomyosin,TM）重组融合蛋白MBP-CTB-TM,将其应用于Balb/c小鼠致敏实验,探究融合蛋白的致敏性及其对小鼠食物过敏相关免疫反应的影响,以达到降低过敏原剂量提高建模效率的效果。结果表明：MBP-CTB-EGFP具有作为黏膜佐剂的能力,从而促进外源蛋白进入HEK293T细胞内,且与转染方式相比,携带外源蛋白进入细胞的效率更高。另一方面,利用融合蛋白MBP-CTB-TM免疫小鼠,TM特异性IgE的OD450 nm达到0.4,而天然TM致敏组仅为0.05,此外融合蛋白致敏还导致高水平的TM特异性IgG1、IgG2a产生。本研究表明,融合蛋白MBP-CTB-TM致敏效果更好,有可能达到降低过敏原用量的效果,为后续食物过敏研究提供了有力工具。     

Bioinformatic screening and detection of allergen cross‐reactive IgE‐binding epitopes

Protein allergens can be related by cross‐reactivity. Allergens that share relevant sequence can cross‐react, those lacking sufficient similarity in their IgE antibody‐binding epitopes do not cross‐react. Cross‐reactivity is based on shared epitopes that is based on shared sequence and higher level structure (charge and shape). Epitopes are important in predicting cross‐reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE‐binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (expectation value, E‐value) was used to identify a threshold specific to identifying cross‐reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross‐reactive or other homologous allergens. A FASTA expectation value range (1 × 10^?5–1 × 10^?6) was identified that could act as a threshold to help identify cross‐reactivity potential.     

碧根果致敏原Car i 1的分离纯化及表征鉴定

目的 分离纯化碧根果致敏原Car i 1,并对其结构进行表征鉴定。方法 以新鲜碧根果果仁为原料,通过粉碎、脱脂、浸提、粗分级、凝胶过滤层析,对碧根果致敏原蛋白Car i 1进行分离纯化。结合十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、液相色谱-串联质谱法和免疫印迹法3种方法对Cari1进行鉴定,并通过圆二色谱仪与紫外分光光度计表征其二、三级结构。结果 本方法纯化获得碧根果致敏原Cari1,单轮制备量可达5 mg以上,且纯度大于95%,蛋白质高级结构未被破坏,能够被全部3名碧根果过敏患者的血清准确识别。结论 该纯化方法技术路线简单、设备要求低且单次制备量高,总得率可达65%,操作便捷,为碧根果致敏原Car i 1的相关研究奠定了物质基础。     

Importance of conformation for the IgE reactivity of sarcoplasmic calcium-binding protein from the black tiger shrimp Penaeus monodon

Sarcoplasmic calcium-binding protein (SCP) is an EF-hand Ca²⁺-binding protein recently identified as a new crustacean allergen. Some EF-hand Ca²⁺-binding allergens, such as parvalbumin (fish allergen) and Bet v 4 (birch pollen allergen), have been shown to contain conformational-type IgE epitopes associated with the Ca²⁺-chelating. This study was performed to clarify the relationships between IgE reactivity and structure of the black tiger shrimp SCP. When analyzed by ELISA in the absence and presence of EGTA, the IgE reactivity of the black tiger shrimp SCP was found to be considerably reduced by Ca²⁺-depletion. Furthermore, circular dichroism spectral data showed a conformational difference between Ca²⁺-bound and Ca²⁺-depleted forms of the black tiger shrimp SCP. On the other hand, the synthetic 18 peptides spanning the entire amino acid sequence of the black tiger shrimp SCP were all assessed to have little IgE-binding ability by ELISA. Our results demonstrate that the IgE reactivity of the black tiger shrimp SCP is attributable mostly to conformational-type IgE epitopes, at least part of which is maintained by Ca²⁺-chelating.     

Purification of Parvalbumin from Carp: A Protocol that Avoids Heat-Treatment

ABSTRACT: Parvalbumin from carp, a major allergen, was purified to homogeneity using ion exchange chromatography and size exclusion chromatography (estimated purity >95% to 98% based on SDS-PAGE and native PAGE) with a yield of 318 mg, and a number of basic biochemical characteristics were determined. The identity was confirmed by peptide-mass fingerprinting, and IgE-binding was demonstrated. The UV/Vis absorbance spectra were explained using the previously published amino acid sequences. Far UV-CD spectroscopy was used to confirm the folding character of parvalbumin. We conclude that parvalbumin from carp can be purified on a comparatively large (hundreds of milligrams) scale using a purification protocol that does not include denaturing steps. The purified protein resembles biochemical characteristics as were earlier published for carp parvalbumin, that is, a molecular weight of approximately 12 kDa, amino acid sequence identity and a secondary structure containing α-helices and β-structures. The described method provides a yield sufficient to produce and characterize antibodies to construct immunochemical methods to detect parvalbumin in food, as well as for use as a standard calibrator for such assays. Practical Application: Parvalbumin is a major allergen from fish. Here, we have purified a comparatively large quantity from carp that can be used to develop antisera for use in an assay method to detect fish allergens.     

Production-scale purification of the recombinant major house dust mite allergen Der f 2 mutant C8/119S

A WHO position paper states that allergen immunotherapy is an effective treatment for allergic diseases, and well characterized allergens should be used in immunotherapy. The house dust mite is a major cause of allergic disease. However, the biological activity of the mite extracts currently used cannot be clearly determined, since these extracts contain various impurities. The use of recombinant allergens can avoid this problem. However, there remains a risk of contamination by other impurities, such as host cell-derived proteins (HCPs). Advanced purification techniques are thus required to remove these contaminants. C8/119S is a mutant of the major house dust mite allergen Der f 2, and is expressed and accumulated as an inclusion body in Escherichia coli. The C8/119S was refolded and purified through three column chromatography steps. Using this method, we could obtain about 2 g of the purified C8/119S in one purification batch. This amount is equivalent to 100,000 of the maintenance doses required for immunotherapy based on the WHO position paper. The purity of the C8/119S was 99% or more. The antigenicity of HCPs in the C8/119S was examined by passive cutaneous anaphylaxis assays. When the C8/119S was administered at 40 μg/kg, no local anaphylaxis was observed. C8/119S was thus highly purified with an extremely low level of impurities, and our procedure was shown to be an effective advanced production-scale purification process for this Der f 2 mutant. In this study, we established an advanced purification processes for C8/119S, then characterized the purified C8/119S and evaluated its purity.     

Reduction of allergenic properties of shrimp (Penaeus Vannamei) allergens by high intensity ultrasound

The tropomyosin fraction of shrimp proteins is potentially responsible for an allergic reaction in individuals with a genetic predisposition to allergy. However, there are no efficient and safe methods to reduce its allergenicity. High-intensity ultrasound is known to change the structure of proteins. The aim of this study was to assess the effect of high-intensity ultrasound on the IgE-binding capacity of shrimp protein extracts (PE) and of major shrimp allergen Pen a 1(tropomyosin). Peeled shrimp (Penaeus vannamei) muscle was sealed in a plastic bag and treated with high-intensity ultrasound (30 Hz, 800 W) for 1.5 h at 0 and 50 °C, respectively; others were treated with boiling water for 15 min. Then PE and Pen a 1 were made with treated shrimp muscle. The allergenicity of PE and purified allergen from different-treated shrimp were analyzed by the enzyme allergosorbent test (EAST) and competitive inhibition ELISA (Ci-ELISA) using sera of 15 shrimp-allergic patients. PE from high-intensity ultrasonic treatment at 50 °C (treated 2) shrimp was 2.2-fold, 2.5-fold lower respectively than that of untreated shrimp and high-intensity ultrasonic treatment at 0 °C (treated 1) shrimp. While PE from heated (treated 3) shrimp was 1.2-fold lower than that of untreated shrimp. The (Concentration of the different-treated sample that inhibit 50% of the IgE binding to coated untreated sample) IC₅₀ ratios of Pen a 1 prepared from treated 2 shrimp, treated 1 shrimp and treated 3 shrimp to untreated shrimp were 1/15, 1/1.1 and 1/1, respectively. The results suggest that high-intensity ultrasound at 50 °C may reduce the allergenicity of shrimp.     

Isolation and characterization of a major allergen in kiwi fruit

 The isolation of an important allergen in kiwi fruit (Actinidia chinensis) by ion-exchange chromatography (IEC) and micropreparative SDS-PAGE followed by electroelution is reported. The purity of the allergen was analysed by SDS-PAGE and immunoblotting with sera from patients who have an allergy to kiwi. The allergen was shown to have a molecular weight of 43 kDa by SDS-PAGE/immunoblotting and an isoelectric point of approximately 6.9 as estimated by IEC. In accordance with World Health Organization nomenclature, this allergen is called Act c 2. By immunoblot inhibition it was shown that epitopes from different allergens in kiwi fruit are also located on Act c 2. N-terminal amino acid sequencing of 17 amino acid residues did not reveal homology with the major allergens in birch pollen (Bet v 1), apple (Mal d 1) or with other proteins of allergenic plant foods. In addition, the isoelectric point of a 67-kDa allergen in kiwi fruit was estimated to be 7.4 by IEC, but micropreparative isolation of this allergen failed because of its very low content in the fruit. Received: 14 April 1997     

Isolation and characterization of a major allergen in kiwi fruit

 The isolation of an important allergen in kiwi fruit (Actinidia chinensis) by ion-exchange chromatography (IEC) and micropreparative SDS-PAGE followed by electroelution is reported. The purity of the allergen was analysed by SDS-PAGE and immunoblotting with sera from patients who have an allergy to kiwi. The allergen was shown to have a molecular weight of 43 kDa by SDS-PAGE/immunoblotting and an isoelectric point of approximately 6.9 as estimated by IEC. In accordance with World Health Organization nomenclature, this allergen is called Act c 2. By immunoblot inhibition it was shown that epitopes from different allergens in kiwi fruit are also located on Act c 2. N-terminal amino acid sequencing of 17 amino acid residues did not reveal homology with the major allergens in birch pollen (Bet v 1), apple (Mal d 1) or with other proteins of allergenic plant foods. In addition, the isoelectric point of a 67-kDa allergen in kiwi fruit was estimated to be 7.4 by IEC, but micropreparative isolation of this allergen failed because of its very low content in the fruit. Received: 14 April 1997     

牛乳中主要过敏原的分离纯化研究进展

牛乳中含有3 种主要的过敏原蛋白：酪蛋白、β- 乳球蛋白和α- 乳白蛋白。本文详细叙述沉淀法、离子交换层析法、凝胶过滤层析法、羟基磷灰石层析法、疏水相互作用层析法、高效液相色谱法以及膜技术等分离纯化技术在牛乳主要过敏原分离纯化中的研究进展。其中,离子交换层析与凝胶层析已被广泛使用,而沉淀法一般作为粗提纯过的步骤。羟基磷灰石层析与疏水相互作用层析法也较为常见,既可单独分离过敏原,又可与其他方法结合来分离过敏原。另外高效液相色谱法与膜技术则是进一步纯化的后续工作,以提高过敏原的纯度。     

可视化膜芯片技术检测常见食源性过敏原成分

目的 建立包括鱼、虾、鸡、鸭、花生、核桃、小麦以及大豆过敏原成分的基因膜芯片技术, 实现对这几种食源性过敏原的同步可视化检测。方法 针对常见食源性过敏物质的成分特异性基因或者过敏原蛋白基因设计特异性的带生物素标记的引物和探针。采用反向斑点杂交(reversedot blot, RDB)结合多重聚合酶链式反应(multiplex polymerase chain reaction, MPCR)技术, 最终通过化学显色直观显示检测结果, 实现对多种食源性过敏原的同步可视化检测。结果 所建立的可视化基因膜芯片方法准确性强, 通过单目标物以及多目标物特异性实验, 显示本方法仅对鱼、虾、花生、核桃、大豆等靶标食源性过敏原有特异性反应, 非靶标物检测均为阴性; 通过制备不同质量浓度比的模拟样品考核方法的灵敏度, 经检测, 方法检测灵敏度可达0.1%(质量分数)。结论 所建立的方法可达到可视化、快速、准确地鉴别食品中鱼、虾、花生、核桃、大豆等常见食源性过敏原。     

南美白对虾主要过敏原原肌球蛋白的低过敏性处理方法研究

以南美白对虾原肌球蛋白(Tropomyosin,Tm)为研究对象,采用酶解法、超声结合酶解法消减3种虾制品中的Tm。首先,以菠萝蛋白酶水解富集的Tm样品通过优化酶活与底物质量比、反应时间和反应温度,建立了能有效消减Tm的酶解方法;同时对比了酶解、超声结合酶解法分别对虾仁、蝴蝶虾仁和虾糜3种制品中的Tm过敏原性变化情况。Tm致敏动物模型的抗血清ELISA结果显示,单纯酶解和超声结合酶解处理的虾仁其过敏原性无显著性差异(P0.05);经超声结合菠萝蛋白酶酶解处理后的蝴蝶虾仁样品其过敏原性降低了21.05%;而虾糜样品中,单纯的酶解或超声结合酶解法与对照相比均有显著性差异,其过敏原性分别减少了30.70%和33.33%。综上所述,作者建立的酶解法可有效消减Tm的过敏原性,该酶解法以及超声结合酶解法在低过敏原性蝴蝶虾仁和虾糜制品的生产领域具有较高的应用可行性。