Tropomyosin (TM) is the main allergen of Macrobrachium nipponense. Recombinant allergens have great prospects in the detection, diagnosis, and treatment of food allergens. The purpose of this study was to compare the differences in structure and allergenicity between natural TM and recombinant TM. Recombinant TM of M. nipponense with a molecular weight of 38 kDa was successfully expressed in the Escherichia coli system. The amino acid sequence as well as secondary structure between natural and recombinant TM were similar, which were verified by mass and CD spectrometry, respectively. Studies showed that both natural TM and recombinant TM had strong allergenicity, and recombinant TM was more allergenic, which could be used as a substitute for natural TM in the diagnosis and treatment of shrimp allergy. This study provided stable and reliable allergen components for the detection of crustacean allergens and the diagnosis and treatment of food allergies caused by crustacean allergens.

BACKGROUND: Recombinant allergens produced by Escherichia coli (E. coli) system play an important role in the component-resolved diagnostics of allergy and vaccine development. However, incorrect folding of recombinant allergens may affect their application. Therefore, it is very important to monitor the correct folding of recombinant allergens. Currently, there is still a lack of a quality control strategy to solve this problem. In this study, a mite allergen, Der f 2, was taken as an example to establish a novel quality control strategy, which was based on chromatography to isolate the allergen, and on enzyme-linked immunosorbent assay to verify the IgE reactivity of the isolated allergen.
METHODS: The nucleotide sequence encoding Der f 2 was codon-optimized and cloned into pET-28a (+) plasmid. Best conditions for the expression of Der f 2 in E. coli were sought. The inclusion body of Der f 2 was denatured and purified by nickel affinity chromatography. Refolding processes were compared using glutathione redox system. The fully and partially folded proteins were separated by anion exchange chromatography, and the IgE reactivity of the isolated proteins was verified by indirect enzyme-linked immunosorbent assay.
RESULTS: An optimized 387 bp segment of the Der f 2 coding gene was successfully expressed in E. coli. Best induction conditions included preinduction bacterial density with absorbance value at 600 nm was 0.6, 1 mM isopropyl beta-d-thiogalactopyranoside at 28°C for 4 h. The Der f 2 protein after refolding was separated by chromatography and two fractions were obtained. The first fraction was identified as monomer protein and the second as aggregate by size-exclusion chromatography. Indirect enzyme-linked immunosorbent assay also confirmed that the first fraction showed higher IgE reactivity.
CONCLUSIONS: In this study, a novel quality control strategy based on chromatographic separation and IgE reactivity monitoring was established in the case of mite Der f 2, which systematically evaluated the effectiveness of multiple preparation methods for the first time. It is faster and more convenient when compared with the existing methods such as size-exclusion chromatography. This strategy laid a foundation for the stable application of recombinant allergens produced by E. coli in component-resolved diagnostics and the development of molecular vaccines in the future.

Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in Spodoptera frugiperda Sf9 cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed Parietaria judaica (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from Helianthus (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.

Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.

Allergen component products, such as recombinant proteins and epitope peptides of allergic components, are used as an adjunct to allergen-specific immunotherapy. We characterized a novel allergen, Tyr p 31, from Tyrophagus putrescentiae, a common allergenic mite. T. putrescentiae total RNA was amplified to Tyr p 31-encoding cDNA, which was inserted into pET28a(+). pET28a(+)-Tyr p 31 was then transformed into Rosetta 2 (DE3) pLysS cells and expressed under isopropyl β-D-thiogalactoside induction. Next, we visualized Tyr p 31 through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting based on its theoretical molecular weight. Recombinant Tyr p 31 (rTyr p 31) was purified, and its secondary structure was noted to comprise α-helices, antiparallel coils, β-turns, parallel coils, and random coils. Our enzyme-linked immunosorbent assay and Western blotting results for T. putrescentiae-positive sera from children with allergic disorders demonstrated rTyr p 31-specific IgE-positivity rates of 72.41 % and 85.7 %, respectively. In BEAS-2B cells, rTyr p 31 increased IL-6 and IL-8 expression; furthermore, BEAS-2B cells treated with 30 μg/mL rTyr p 31 demonstrated 100 upregulated and 12 downregulated genes. In summary, we identified Tyr p 31, a novel T. putrescentiae allergen component, and noted rTyr p 31 to have a high IgE-binding rate and strong immunogenicity.

Banana allergy is often associated with the pollen and latex allergies, which led us to the hypothesis that some yet unidentified banana allergen could provide a basis of the latex-pollen-fruit syndrome. S-adenosyl-l-homocysteine hydrolase (SAHH) was recently identified in the literature as a novel plant allergen. This study aimed to assess the allergenic potential of the naturally occurring banana SAHH (nSAHH) and its recombinant homolog produced in E. coli (rSAHH). nSAHH showed IgE reactivity with a serum pool of twelve banana-allergic persons, while rSAHH displayed IgE reactivity in ten out of the twelve tested patients. Five linear B-cell epitopes were identified on the rSAHH surface, exhibiting ≥ 90 % sequence homology with relevant plant SAHH allergens. Our findings have elucidated SAHH as a novel plant panallergen, underlying the cross-reactivity between plant-derived food and respiratory allergens, confirming our initial hypothesis.

The methylotrophic yeast Pichia pastoris (P. pastoris) is one of the expression systems widely used to produce recombinant heterologous proteins. In this chapter, the methodology to produce recombinant food allergens in P. pastoris is described. The methodology begins with the preparation of competent P. pastoris cells followed by the transformation of the competent cells by electroporation as well as the preparation of plasmid DNA for transformation. Moreover, the screening of yeast transformants by direct PCR to ensure integration of allergen DNA followed by small-scale expression of recombinant allergen in yeast cells is also described.

Shellfish are a leading cause of allergies worldwide, affecting about one-tenth of the general population. The sarcoplasmic calcium-binding protein, also known as allergen Pen m 4, is an important factor in shrimp allergies. Our objective was to assess the most effective techniques for producing a recombinant Pen m 4 protein as a potential tool for diagnosing shrimp allergies. In this study, for the first time, we produced a functional recombinant Pen m 4 protein in a eukaryotic system, Pichia pastoris, and analyzed it against Escherichia coli-produced equivalents in enzyme-linked immunosorbent and reverse-phase protein microarray assays. A dual tag system based on the maltose-binding protein was successfully used to increase the yield of Pen m 4 by 1.3-2.3-fold in both bacteria and yeast, respectively. Immunological characterization showed that N-glycosylation is neither crucial for the folding of Pen m 4 nor its recognition by specific IgE. However, the Ca2+-depletion assay indicated a dependence on calcium ion presence in blood samples. Results demonstrate how a comparative analysis can elucidate essential allergen manufacturing points. In conclusion, E. coli-produced Pen m 4 protein fused with the maltose-binding protein should be the preferred option for further studies in Penaeus monodon allergy diagnostics.

House dust mite (HDM) allergy belongs to the most important allergies and affects approximately 65-130 million people worldwide. Additionally, untreated HDM allergy may lead to the development of severe disease manifestations such as atopic dermatitis or asthma. Diagnosis and immunotherapy of HDM allergic patients are well established but are often hampered by the use of mite extracts that are of bad quality and lack important allergens. The use of individual allergens seems to be a promising alternative to natural allergen extracts, since they represent well-defined components that can easily be produced and quantified. However, a thorough characterization of the individual allergens is required to determine their clinical relevance and to identify those allergens that are required for correct diagnosis of HDM allergy and for successful immunotherapy. This review gives an update on the individual HDM allergens and their benefits for diagnosis and immunotherapy of HDM allergic patients.

Exposure to the Mus m 1 aeroallergen is a significant risk factor for laboratory animal allergy. This allergen, primarily expressed in mouse urine where it is characterized by a marked and dynamic polymorphism, is also present in epithelium and dander. Considering the relevance of sequence/structure assessment in protein antigenic reactivity, we compared the sequence of the variant Mus m 1.0102 to other members of the Mus m 1 allergen, and used Discotope 2.0 to predict conformational epitopes based on its 3D-structure. Conventional diagnosis of mouse allergy is based on serum IgE testing, using an epithelial extract as the antigen source. Given the heterogeneous and variable composition of extracts, we developed an indirect ELISA assay based on the recombinant component Mus m 1.0102. The assay performed with adequate precision and reasonable diagnostic accuracy (AUC = 0.87) compared to a routine clinical diagnostic test that exploits the native allergen. Recombinant Mus m 1.0102 turned out to be a valuable tool to study the fine epitope mapping of specific IgE reactivity to the major allergen responsible for mouse allergy. We believe that advancing in its functional characterization will lead to the standardization of murine lipocalins and to the development of allergen-specific immunotherapy.