Efficient soluble production of folded cat allergen Fel d 1 in Escherichia coli.

The major cat allergen Fel d 1 is one of the most common and potent causes of animal related allergy. Medical treatment of cat allergy has relied on immunotherapy carried out with cat dander extract. This approach has been problematic, mainly due to inconsistent levels of the major allergen in the produced extracts. Recombinant DNA technology has been proposed as an alternative method to produce more consistent pharmaceuticals for immunotherapy and diagnostics of allergy. Current approaches to produce recombinant Fel d 1 (recFel d 1) in the cytoplasm of Escherichia coli have however resulted in protein folding deficiencies and insoluble inclusion body formation, requiring elaborate in vitro processing to acquire folded material. In this study, we introduce an efficient method for cytoplasmic production of recFel d 1 that utilizes eukaryotic folding factors to aid recFel d 1 to fold and be produced in the soluble fraction of E. coli. The solubly expressed recFel d 1 is shown by biophysical in vitro experiments to contain structural disulfides, is extremely stable, and has a sensitivity for methionine sulfoxidation. The latter is discussed in the context of functional relevance.

Cloning, expression, and purification of recombinant major mango allergen Man i 1 in Escherichia coli.

In recent years, the number of people around the world who suffer from fruit allergies has increased. Mango can induce anaphylaxis, and two major mango allergens have been identified - Man i 1 and Man i 2. Apart from their molecular weights and pI values, no other information about them is known. This work identifies the DNA and amino acid sequences of Man i 1 and constructs an expression system for recombinant Man i 1 (rMan i 1). Firstly, 3' and 5' RACE assays were used to identify the cDNA fragment of Man i 1. Subsequently, the full length of Man i 1 cDNA was inserted into a pET-21a(+) vector, and the inserted plasmid was transformed to Escherichia coli BL21 (DE3) to express rMan i 1. The conditions for the expression of rMan i 1, including IPTG concentration, induction temperature, and induction time, were optimized. The highest amount of soluble rMan i 1 was obtained after induction with 0.1 mM IPTG at 16 °C for 20 h. The His-tagged rMan i 1 was purified using Ni-NTA agarose and its identity was verified using an anti-histidine antibody and the serum of a mango-allergic person. Additionally, rMan i 1 was identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and shared 86.2% identity in amino acid sequence of GAPDH from wheat. Finally, an E. coli expression system of rMan i 1 was established, with the potential to be used in immunotherapy against mango allergy or the development of assays for detecting the residues of mango allergens.

Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli.

Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.

The Innovation from Innsbruck: Plant-based Allergen Recombinantly Produced in Green Alga.

An innovation in biotechnology: for the first time, researchers from MCI Innsbruck and the University of Salzburg have manufactured and purified a plant-based allergen in a green alga and opened the door to a specific immunotherapy against allergies. Their vision: to replace the unpleasant injection with oral administration, as its production is both simple and cost-effective.

Production and analysis of recombinant tree nut allergens.

Allergic reactions to tree nuts are a growing global concern as the number of affected individuals continues to rise. Unlike some food allergies, tree nuts can cause severe reactions that persist throughout life. The tree nuts discussed in this review include those most commonly responsible for allergic reactions: cashew, almond, hazelnut, walnut, pecan, Brazil nut, pistachio, and chestnut. The native allergenic proteins derived from tree nuts are frequently difficult to isolate and purify and may not be adequately represented in aqueous nut protein extracts. Consequently, defined recombinant allergens have become useful reagents in a variety of immunoassays aimed at the diagnosis of tree nut allergy, assessing cross-reactivity between various nuts and other seeds, mapping of IgE binding epitopes, and analyzing the effects of the food matrix, food processing, and gastric digestion on allergenicity. This review describes the approaches that can be used for the production of recombinant tree nut allergens and addresses key issues associated with their production and downstream applications.

The site of allergen expression in hematopoietic cells determines the degree and quality of tolerance induced through molecular chimerism.

The transplantation of allergens (e.g. Phl p 5 or Bet v 1) expressed on BM cells as membrane-anchored full-length proteins leads to permanent tolerance at the T-cell, B-cell, and effector-cell levels. Since the exposure of complete allergens bears the risk of inducing anaphylaxis, we investigated here whether expression of Phl p 5 in the cytoplasm (rather than on the cell surface) is sufficient for tolerance induction. Transplantation of BALB/c BM retrovirally transduced to express Phl p 5 in the cytoplasm led to stable and durable molecular chimerism in syngeneic recipients (∼20% chimerism at 6 months). Chimeras showed allergen-specific T-cell hyporesponsiveness. Further, Phl p 5-specific TH 1-dependent humoral responses were tolerized in several chimeras. Surprisingly, Phl p 5-specific IgE and IgG1 levels were significantly reduced but still detectable in sera of chimeric mice, indicating incomplete B-cell tolerance. No Phl p 5-specific sIgM developed in cytoplasmic chimeras, which is in marked contrast to mice transplanted with BM expressing membrane-anchored Phl p 5. Thus, the expression site of the allergen substantially influences the degree and quality of tolerance achieved with molecular chimerism in IgE-mediated allergy.

Characterization of the house dust mite allergen Der p 21 produced in Pichia pastoris.

BACKGROUND: The development of recombinant house dust mite (HDM) allergens opened the way for the in-depth characterization of these molecules but also provided new opportunities to refine the diagnostic procedures of HDM allergy as well as the allergen-specific immunotherapy through tailor-made treatments. OBJECTIVE: In the present study, the HDM allergen Der p 21 was expressed in Pichia pastoris under a secreted form. The physico-chemical as well as the allergenic characterizations of recombinant Der p 21 (rDer p 21) were performed. METHODS: Purified rDer p 21, secreted from recombinant P. pastoris was characterized by CD and MS analysis and the frequency of IgE reactivity was determined by ELISA using 96 sera of HDM-allergic patients from Bangkok. The direct airway epithelial cell activation by rDer p 21 was also evaluated. RESULTS: rDer p 21 was highly expressed under a secreted form in P. pastoris. The physico-chemical characterization of purified rDer p 21 showed that the allergen displayed appropriate α-helix secondary structure content although a two amino acids truncation at the N-terminus of the protein was evidenced by MS. The prevalence of IgE reactivity to rDer p 21 reached 25% in the cohort of the HDM-allergic patients. rDer p 21 could trigger IL-8 production in airway epithelial cells through TLR2-dependent signaling. CONCLUSION: Properly folded rDer p 21 produced in P. pastoris is appropriate for HDM allergy diagnosis as well for future recombinant allergen-based specific immunotherapy.

Antibodies generated against conserved antigens expressed by bacteria and allergen-bearing fungi suppress airway disease.

There has been a sharp rise in allergic asthma and asthma-related deaths in the developed world, in contrast to many childhood illnesses that have been reduced or eliminated. The hygiene hypothesis proposes that excessively sanitary conditions early in life result in autoimmune and allergic phenomena because of a failure of the immune system to receive proper microbial stimulation during development. We demonstrate that Abs generated against conserved bacterial polysaccharides are reactive with and dampen the immune response against chitin and Aspergillus fumigatus. A reduction in Ag uptake, cell influx, cell activation, and cytokine production occurred in the presence of anti-polysaccharide Abs, resulting in a striking decrease in the severity of allergic airway disease in mice. Overall, our results suggest that Ag exposure--derived from environmental sources, self-antigens, or vaccination--during the neonatal period has dramatic effects on the adult Ab response and modifies the development of allergic airway disease.

Thymic stromal lymphopoietin is produced by dendritic cells.

Thymic stromal lymphopoietin (TSLP) is a type 1 cytokine that contributes to lymphopoiesis and the development of asthma and atopic dermatitis. TSLP acts on multiple lineages, including dendritic cells (DCs), T cells, NKT cells, eosinophils, and mast cells, mediating proliferation and survival and linking innate and adaptive immune responses. TSLP is produced by a range of cells, including epithelial cells, fibroblasts, stromal cells, and keratinocytes. DCs are important primary targets of TSLP, and we unexpectedly demonstrated that DCs also produce TSLP in response to TLR stimulation and that this is augmented by IL-4. Moreover, we demonstrated that when mice were challenged with house dust mite extract, lung CD11c(+) DCs expressed TSLP mRNA at an even higher level than did epithelial cells. These data suggested that DCs not only respond to TSLP but also are a source of TSLP during pathogen and/or allergen encounter.

Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis.

Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.

The advent of recombinant allergens and allergen cloning.

When the allergen nomenclature system was adopted in 1986, allergens were identified by their behavior on electrophoresis and chromatography and by reactivity to shared antisera. Not only was this unsatisfactory for standardization, but the processes of allergic sensitization and immunotherapy could not be studied in the framework of antigen processing and B- and T-cell epitopes. Recombinant technologies developed in the 1980s for cloning cDNA from low-abundance mRNA permitted the cloning of allergens, beginning with the major house dust mite allergen Der p 1 and hornet allergen Dol m 5. After this, a wave of cloning with IgE immunoscreening resulted in the cloning of Der p 2, Der p 5, Bet v 1, Bet v 2, and Dac g 2 along with Fel d 1 cloned after amino acid sequencing. Recombinant allergens have now been used to define the important allergens for a wide range of allergies and to develop new types of immunotherapy, some of which have shown efficacy in human trials. The clonally pure allergens have been used to solve the tertiary structures of allergens and from this how allergens might activate innate immunity. Proprietary recombinant allergens are now being used in improved diagnostic tests.

Synthesis and degradation of the major allergens in developing and germinating soybean seed.

Gly m Bd 28K, Gly m Bd 30K and Gly m Bd 60K are the major soybean (Glycine max (L.) Merr.) allergens limiting the consumption of a good protein source for sensitive individuals. However, little is known about their temporal-spatial expression during seed development and upon germination. The present data shows that soy allergens accumulated in both the embryonic axes and cotyledon, but expression patterns differed depending on the specific allergen. Allergens accumulated sooner and to a greater level in cotyledons than in embryonic axes. Gly m Bd 28 began at 14 d after flowering, 7 to 14 d earlier than Gly m Bd 30K and Gly m Bd 60K. Comparatively, their degradation was faster and more profound in embryonic axes than in cotyledons. Gly m Bd 60K began to decline at 36 h after imbibition and remained detectable up to 108 h in cotyledons. In contrast, the Glym Bd 60K protein was reduced at 24 h, and eventually disappeared at 96 h . In cotyledons Gly m Bd 28K first declined at 24 h, then increased from 36 h to 48 h, followed by its large reduction at 72 h after seed germination. These findings provide useful information on soy allergen biosynthesis and will help move forward towards developing a hypoallergenic soybean for safer food.

Expression of a major plant allergen as membrane-anchored and secreted protein in human cells with preserved T cell and B cell epitopes.

BACKGROUND: Expression of allergens in human cells is a prerequisite for the development of antigen-specific cell therapy in IgE-mediated allergy. We developed a strategy how the clinically relevant major grass pollen allergen Phl p 5 can be efficiently secreted or expressed on the surface of human cells with preserved allergenic activity. METHODS: The cDNA of Phl p 5 was fused to a leader peptide with or without a transmembrane domain and both constructs were ligated into a mammalian expression vector. Transfection of these plasmids into human cells resulted in a membrane-anchored or secreted version of Phl p 5, respectively, as determined by ELISA or flow cytometric analysis. RESULTS: Both the secreted and membrane-anchored Phl p 5 proteins bound IgE from allergic patients in an immunoblot assay and induced specific histamine release and CD203c upregulation in basophils of grass pollen-allergic patients. Proliferation of peripheral blood mononuclear cells from Phl p 5-allergic individuals was induced upon stimulation with both variants of Phl p 5 expressed in human cells similar to recombinant Phl p 5. CONCLUSIONS: Secreted and membrane-anchored Phl p 5 expressed in human cells preserved B cell as well as T cell epitopes and may be used to develop and test various cell-based strategies for allergen-specific immunomodulation and to delineate the tolerance mechanisms involved therein.

Distribution of core fragments from the major cat allergen Fel d 1 is maintained among the main anatomical sites of production.

BACKGROUND: Polymorphism of Fel d 1 has long been observed, but structural characterization of Fel d 1 variants among the various sites of production and animals is still missing. This study was aimed at elucidating the structural polymorphism of this protein as a function of the site of production and the resulting influence on the immunological behavior of the allergen. METHODS: Fel d 1 was water-extracted from the 4 main sites of production, i.e. cheek zone, interdigital zone, anal sac (AS), and chest area and a panel of 10 cats. Fel d 1-like materials were immunoblotted, immunopurified and characterized by MALDI-TOF MS. Immunoreactivity was studied using ELISA dose-dependent curves at equilibrium. RESULTS: In all areas, 4-10 isoforms ranging from 7 to 40 kDa were detected by MS. Essentially two truncated heterodimers of 13-14 and 16-17 kDa were identified together with intact Fel d 1 in all compartments and they were largely predominant in AS. These core fragments were found to contribute to a variable recognition by antibodies in a panel of ELISA. CONCLUSIONS: Our study identified truncated dimers of Fel d 1, probably resulting from proteolytic degradation of both chains and present in all cats. Core fragments are largely distributed among anatomical sites of production and especially well represented in AS. They are recognized as intact allergens by antibodies and may therefore introduce discrepancies in allergen measurement depending on the variable amount of intact and degraded Fel d 1 produced by the cat.

Pichia pastoris is superior to E. coli for the production of recombinant allergenic non-specific lipid-transfer proteins.

Non-specific lipid-transfer proteins (nsLTP) from food and pollen are clinically important allergens, especially in patients recruited from the Mediterranean area. For the use of recombinant nsLTPs in allergy diagnosis and preclinical allergy studies the preparation of nsLTPs in a properly folded and biologically active form is required. Using hazelnut nsLTP (Cor a 8) as a model allergen, heterologous over-expression in Escherichia coli and Pichia pastoris was compared. Recombinant Cor a 8 derived from E. coli and P. pastoris was purified by IMAC and SEC or ammonium sulphate precipitation followed by IEC and SEC, respectively. The recombinant proteins were characterized with regard to IgE-binding by immunoblotting and ELISA, structure by N-terminal sequencing, CD-spectroscopy and LS and to their biological activity using an in vitro basophil histamine release assay. Purification of hazelnut nsLTP from bacterial lysate under native conditions resulted in a low yield of Cor a 8. In addition, the preparation contained non-IgE-reactive aggregations besides the IgE-reactive monomer. In contrast, the yield of rCor a 8 produced in P. pastoris was approximately 270-fold higher and impurities with oligomers have not been detected. Purified monomeric Cor a 8 from bacteria and yeast showed similar IgE-antibody reactivity and secondary structures, and both were capable of inducing histamine release from basophils. In summary, P. pastoris is superior to E. coli as expression system for the production of large quantities of soluble, properly folded, and biologically active rCor a 8.

Cloning, expression, characterization, and computational approach for cross-reactivity prediction of manganese superoxide dismutase allergen from pistachio nut.

BACKGROUND: Tree nut allergy is one of the common potentially life-threatening food allergies in children and adults. Recombinant food allergens offer new perspectives to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of food allergies. So far, superoxide dismutase (s) has been identified as a panallergen and studied in different allergenic sources. Manganese Superoxide Dismutase (MnSOD) has also been reported in pistachio that may cause allergic reactions in atopic subjects. The aim of this study was to describe the cloning, expression, and purification of MnSOD from pistachio nut. METHODS: The pistachio MnSOD was cloned and expressed in E. coli BL21 (DE3) using a vector pET-32b (+). A recombinant protein was purified by metal precipitation. The protein immunoreactivity was evaluated using patients' IgE binding by means of ELISA and immunoblotting assays. RESULTS: The MnSOD gene from pistachio was successfully cloned and expressed in E. coli. The purified pistachio MnSOD was recognized by IgE in 10 (40%) out of the 25 sera tested. Our results also showed that this protein might trigger some cross-reactions toward IgE antibodies and thus could be considered as a panallergen. CONCLUSIONS: For the first time recombinant manganese superoxide dismutase from nut source was expressed as a possible allergen. This pistachio allergen could be a possible basis for cross-reactivity with MnSOD from other sources.

Design, production and immunomodulatory potency of a novel allergen bioparticle.

Allergen immunotherapy (AIT) is the only disease-modifying treatment with evidence for sustained efficacy. However, it is poorly developed compared to symptomatic drugs. The main reasons come from treatment duration implying monthly injections during 3 to 5 years or daily sublingual use, and the risk of allergic side-effects. To become a more attractive alternative to lifelong symptomatic drug use, improvements to AIT are needed. Among the most promising new immunotherapy strategies is the use of bioparticles for the presentation of target antigen to the immune system as they can elicit strong T cell and B cell immune responses. Virus-like particles (VLPs) are a specific class of bioparticles in which the structural and immunogenic constituents are from viral origin. However, VLPs are ill-suited for use in AIT as their antigenicity is linked to structure. Recently, synthetic biology has been used to produce artificial modular bioparticles, in which supramolecular assemblies are made of elements from heterogeneous biological sources promoting the design and use of in vivo-assembling enveloped bioparticles for viral and non-viral antigens presentation. We have used a coiled-coil hybrid assembly for the design of an enveloped bioparticle (eBP) that present trimers of the Der p 2 allergen at its surface, This bioparticle was produced as recombinant and in vivo assembled eBPs in plant. This allergen biotherapeutic was used to demonstrate i) the capacity of plants to produce synthetic supramolecular allergen bioparticles, and ii) the immunomodulatory potential of naturally-assembled allergen bioparticles. Our results show that allergens exposed on eBPs induced a very strong IgG response consisting predominantly of IgG2a in favor of the TH1 response. Finally, our results demonstrate that rDer p 2 present on the surface of BPs show a very limited potential to stimulate the basophil degranulation of patient allergic to this allergen which is predictive of a high safety potential.

Identification and in silico bioinformatics analysis of PR10 proteins in cashew nut.

Proteins from cashew nut can elicit mild to severe allergic reactions. Three allergenic proteins have already been identified, and it is expected that additional allergens are present in cashew nut. pathogenesis-related protein 10 (PR10) allergens from pollen have been found to elicit similar allergic reactions as those from nuts and seeds. Therefore, we investigated the presence of PR10 genes in cashew nut. Using RNA-seq analysis, we were able to identify several PR10-like transcripts in cashew nut and clone six putative PR10 genes. In addition, PR10 protein expression in raw cashew nuts was confirmed by immunoblotting and liquid chromatography-mass spectrometry (LC-MS/MS) analyses. An in silico allergenicity assessment suggested that all identified cashew PR10 proteins are potentially allergenic and may represent three different isoallergens.