Expression and analysis of recombinant Amb a V and Amb t V allergens. Comparison with native proteins by immunological assays and NMR spectroscopy.

The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response. We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins. Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies. Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells. Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E. coli.     

Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli

The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni²⁺ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more -helix and less -structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.     

Molecular cloning and characterization of Cup a 4, a new allergen from Cupressus arizonica

Sensitization to Cupressaceae pollen has become one of the most important causes of pollinosis in Western countries during winter and early spring. However, the characterization of the extracts, the allergens involved and the cross-reactivity with other pollen sources still remain poorly studied; in the case of Cupressus arizonica only two allergens have been described so far. A new allergen from C. arizonica pollen, Cup a 4, was cloned and expressed in Escherichia coli as an N-terminally His-tag recombinant protein that was characterized biochemically, immunologically and by circular dichroism spectroscopy. The new allergen has high sequence identity with Prickly Juniper allergen Jun o 4 and contains four EF-hand domains. The recombinant protein has structural similarities with other calcium binding allergens such as Ole e 3, Ole e 8 and Phl p 7. Cup a 4 is expressed in mature pollen grains and shares antigenic properties with the recombinant form. Sera from 9.6% C. arizonica allergic patients contain specific IgE antibodies against recombinant Cup a 4.     

Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.     

Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry

The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.     

Ca2+-binding allergens from olive pollen exhibit biochemical and immunological activity when expressed in stable transgenic Arabidopsis

Employing transgenic plants as alternative systems to the conventional Escherichia coli, Pichia pastoris or baculovirus hosts to produce recombinant allergens may offer the possibility of having available edible vaccines in the near future. In this study, two EF-hand-type Ca2+-binding allergens from olive pollen, Ole e 3 and Ole e 8, were produced in transgenic Arabidopsis thaliana plants. The corresponding cDNAs, under the control of the constitutive CaMV 35S promoter, were stably incorporated into the Arabidopsis genome and encoded recombinant proteins, AtOle e 3 and AtOle e 8, which exhibited the molecular properties (i.e. MS analyses and CD spectra) of their olive and/or E. coli counterparts. Calcium-binding assays, which were carried out to assess the biochemical activity of AtOle e 3 and AtOle e 8, gave positive results. In addition, their mobilities on SDS/PAGE were according to the conformational changes derived from their Ca2+-binding capability. The immunological behaviour of Arabidopsis-expressed proteins was equivalent to that of the natural- and/or E. coli-derived allergens, as shown by their ability to bind allergen-specific rabbit IgG antiserum and IgE from sensitized patients. These results indicate that transgenic plants constitute a valid alternative to obtain allergens with structural and immunological integrity not only for scaling up production, but also to develop new kind of vaccines for human utilization.     

Recombinant allergens for analysing T-cell responses

T-cell responses constitute a central element of allergic disease and a model for studying Th1 and Th2 cytokine pathways. Most studies to date have used extracts of allergens which contain variable quantities of different allergens and non-allergenic antigens. Recombinant allergens provide the tools for studying the responses to allergens in a reproducible and dose-dependent manner and the different T-cell responses of allergic and non-allergic subjects provide a method for verifying the responses and their relationship to allergic sensitisation. Most allergies show dominant responses to one or a few major allergens. These allergens have been described for the common allergies and have been produced as recombinant allergens. A particular problem for allergens is that many are mixtures of proteins from multi-gene families or are highly polymorphic. Information now exists so the sequence variation can be represented. Purified recombinant allergens produced by standard expression systems stimulate the expected T-cell responses from the peripheral blood of allergic and non-allergics to allergen extracts. Although stimulation with recombinant allergens which are not produced with a natural IgE binding activity can provide a measure of allergenicity, the altered tertiary structure can reduce Th2 responses. The sequence information now available provides the means to use PCR to produce cDNA for the production of recombinant allergens from readily available sources. The production of the highly reactive recombinant Der p 2 allergen of house dust mite from natural sources is described.     

Native and recombinant proguanylin feature identical biophysical properties and are monomeric in solution

Guanylin, an intestinal peptide hormone and endogenous ligand of guanylyl cyclase C, is produced as the corresponding prohormone proguanylin. The mature hormone consists of 15 amino acid residues, representing the COOH-terminal part of the prohormone comprised of 94 amino acid residues. Here we report the recombinant expression and purification of proguanylin with its native disulfide connectivity, as well as the biophysical characterization of the recombinant and native protein. The comparison of recombinant and native proguanylin revealed identical biophysical and structural properties, as deduced from CZE, HPLC, and mass spectrometry, as well as NMR spectroscopy and CD spectroscopy at various temperatures and pH values. Exhaustive analytical ultracentrifugation studies were employed for protein concentrations up to the millimolar range to determine the association state of recombinant as well as native proguanylin, revealing both proteins to be monomeric at the applied solution conditions. As a result, a former identified close proximity between the termini of proguanylin is due to intramolecular interactions.     

High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies

The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure.     

Over-expression and production of plant allergens by molecular farming strategies

Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future. To supply the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by molecular farming is an alternative to microbial expression systems. Especially as post-translational modifications of the allergens, e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen. An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in this paper.     

Hypoallergenic derivatives of major grass pollen allergens for allergy vaccination

Grass pollen-induced hay-fever and allergic asthma represent a major health problem in industrialized countries. Whereas the symptoms of these allergic conditions can be controlled by pharmacotherapy, specific immunotherapy vaccination is the only causative approach towards the treatment of these type 1 allergies. Specific immunotherapy is based on administration of increasing amounts of the disease-causing allergens in the form of allergen-containing extracts. However, the extracts used for immunotherapy consist of allergenic and non-allergenic components and may induce severe anaphylactic side-effects upon therapeutic administration. With recent developments in molecular biology of pollen allergens it has become feasible to produce modified hypoallergenic derivatives of recombinant allergens with abrogated or greatly reduced likelihood of anaphylactic side-effects as compared to extract-based treatments. We have demonstrated this concept through reducing the anaphylactic potential of major rye grass pollen allergens by introducing a few point mutations which leave the overall structural fold of the molecule unaltered. These modified forms are expected to make allergen-specific immunotherapy more widely used in the future.     

Structural and immunologic characterization of Ara h 1, a major peanut allergen

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170-586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.     

Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.     

Microarrayed allergens for IgE profiling

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.     

Mosquito allergy and mosquito salivary allergens

Allergic reactions to mosquito bites are caused by allergens in mosquito saliva. In this review, allergic reactions to mosquito salivary allergens, and characteristics of salivary allergens and their recombinant forms are described. The use of the recombinant allergens in the diagnosis of mosquito allergy is discussed.     

Provocation testing with recombinant allergens

In the past few decades, DNA technology has enabled the production of defined recombinant allergen molecules for diagnostic and therapeutic purposes. Recombinant allergens containing most of the relevant IgE epitopes present in natural allergen sources are now available and allergen proteins can be produced that are identical, without biological or batch-to-batch variation. A great advantage of recombinant allergens is that they can be used for component-resolved diagnostics, which makes it possible to establish the patient's individual IgE reactivity profile before therapy is selected. However, before recombinant allergens can be applied in clinical practice their biological activity has to be carefully investigated in vivo. We here describe the most commonly used provocation methods (skin tests (prick and intradermal), nasal, bronchial, and conjunctival provocations) and how they can be performed. We also discuss the results so far obtained with in vivo testing using recombinant allergens and envisage their future use for immunotherapy.     

Structure study of recombinant RGD-hirudin by vibrational and circular dichroism spectroscopy

The secondary structure of a new type of recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, has been studied by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy and circular dichroism (CD) methods. The distribution of various secondary structure elements was determined using only a very small amount of sample protein. It was found that the recombinant RGD-hirudin contains about 26% extended chain, 21% beta-turn and 53% unordered structure, leaving no alpha-helix. The results showed that the regular secondary structure of recombinant RGD-hirudin is increased compared with wild-type hirudin. The RGD segment that is located at the end of a long arm of a beta-sheet is thought to play an important role in the additional function of anti-platelet aggregation. Throughout the experiments, FT-IR, Raman spectroscopy and CD generated mutually reinforcing results.     

Circular dichroism and guanine quadruplexes

Circular dichroism (CD) is remarkably sensitive to the conformational states of nucleic acids; therefore, CD spectroscopy has been used to study most features of DNA and RNA structures. Quadruplexes are among the significant noncanonical nucleic acids architectures that have received special attentions recently. This article presents examples on the contribution of CD spectroscopy to our knowledge of quadruplex structures and their polymorphism. The examples were selected to demonstrate the potential of this simple method in the quadruplex field. As CD spectroscopy detects only the global feature of a macromolecule, it should preferably be used in combination with other techniques. On the other hand, CD spectroscopy, often as a pioneering approach, can reveal the formation of particular structural arrangements, to search for the conditions stabilizing the structures, to follow the transitions between various structural states, to explore kinetics of their appearance, to determine thermodynamic parameters and also detect formation of higher order structures. This article aims to show that CD spectroscopy is an important complementary technique to NMR spectroscopy and X-ray diffraction in quadruplex studies.     

Strategies for converting allergens into hypoallergenic vaccine candidates

Specific immunotherapy is based on the administration of increasing doses of allergens to allergic patients with the aim of inducing a state of antigen-specific unresponsiveness. Specific immunotherapy is one of the few causative treatment approaches for Type I allergy but may cause numerous side effects, including local inflammatory reactions, systemic manifestations (e.g., asthma attacks) and in the worst case, anaphylactic shock which may lead to death. Several attempts have been made in the past to reduce the rate of side effects. They included the chemical modification of allergen extracts to reduce their allergenic activity and the adsorption of allergen extracts to adjuvants to prevent the systemic release of allergens after administration. During the last decade, cDNAs coding for the most relevant allergens have been isolated and the corresponding allergens have been produced as recombinant molecules. Using allergen-encoding cDNAs, the amino acid sequence of allergens or purified recombinant allergens several strategies can now be applied to produce allergen derivatives with reduced allergenic activity for allergy vaccination in a controlled and reproducible manner. Currently, allergen-encoding cDNAs are used to engineer recombinant hypoallergenic allergen derivatives. According to the amino acid sequences and experimental epitope mapping data, synthetic peptides representing T- or B-cell epitopes are produced and purified recombinant allergens are coupled to novel adjuvants for vaccine formulation. In this article, strategies for the production and evaluation of allergen derivatives with reduced allergenic activity for allergy vaccination are described. These new vaccines hold great promise to improve the current practice of allergen-specific immunotherapy and maybe also used for prophylactic vaccination in the future.