In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.
Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.
1D 1H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D 1H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins.
Hazelnut is reported as a causative agent of allergic reactions. However it is also an edible nut with health benefits. The allergenic characteristics of hazelnut-samples after autoclaving (AC) and high-pressure (HHP) processing have been studied and are also presented here. Previous studies demonstrated that AC treatments were responsible for structural transformation of protein structure motifs. Thus, structural analyses of allergen proteins from hazelnut were carried out to observe what is occurring in relation to the specific-IgE recognition of the related allergenic proteins. The aims of this work are to evaluate the effect of AC and HHP processing on hazelnut in vitro allergenicity using human-sera and to analyse the complexity of hazelnut allergen-protein structures.
Hazelnut-samples were subjected to AC and HHP processing. The specific IgE- reactivity was studied in 15 allergic clinic-patients via western blotting analyses. A series of homology-based-bioinformatics 3D-models (Cora 1, Cora 8, Cora 9 and Cora 11) were generated for the antigens included in the study to analyse the co mplexity of their protein structure. This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.
A severe reduction in vitro in allergenicity to hazelnut after AC processing was observed in the allergic clinic-patients studied. The specific-IgE binding of some of the described immunoreactive hazelnut protein-bands: Cora 1 ~18KDa, Cora 8 ~9KDa, Cora 9 ~35-40KDa and Cora 11 ~47-48 KDa decreases. Furthermore a relevant glycosylation was assigned and visualized via structural analysis of proteins (3D-modelling) for the first time in the protein-allergen Cora 11 showing a new role which could open a new door for allergenicity-unravellings.
Hazelnut allergenicity-studies in vivo via Prick-Prick and other means using AC processing are crucial to verify the data we observed via in vitro analyses. Glycosylation studies provided us with clues to elucidate, in the near future, mechanisms of the structures that contribute to hazelnut allergenicity, which thus, in turn, help alleviate food allergens.
Structural analysis of allergen-proteins and Glycosylation
Clinically it is recognized that tree nut allergies such as hazelnut allergy are not usually outgrown. Specific mechanisms underlying the persistence of such food allergies are incompletely understood. Here we studied the natural history and the long-term immune and clinical characteristics of hazelnut allergy in an adjuvant-free mouse model.
BALB/c mice were sensitized to hazelnut protein using a transdermal sensitization protocol that does not use adjuvant. After establishing sensitization, exposure to hazelnut was withdrawn for 3, 5 or 8 months. The fate of circulating IgE antibodies was monitored. Subsequently, mice were given booster exposures and examined for memory IgE antibody and spleen cell IL-4 responses. Clinical characteristics and hypothermia responses upon oral allergen challenge were studied.
Upon allergen withdrawal, circulating hazelnut-specific IgE antibody levels began to drop. Nevertheless, IgE responses once established remained at significantly high levels for up to 8 months (the last time point studied) despite withdrawal of allergen exposure. Memory IgE responses to booster exposures were robust after 3, 5 or 8 months of allergen withdrawal. Furthermore, significant clinical reactivity to oral hazelnut challenge, and hypothermia responses were demonstrable at each of these time points. Long-lasting spleen cell memory IL-4 responses to hazelnut were detectable in these mice explaining the mechanism of sustenance of IgE responses and clinical sensitization.
Hazelnut allergy once established persists for long periods, despite withdrawal of allergen exposure, due to long-lasting, memory IgE and IL-4 responses.
Hazelnut allergy, natural history; Systemic anaphylaxis; Immunoglobulin E; T helper 2 lymphocyte profile; Mouse model, adjuvant-free; Food allergy
The crystallization of the brazil nut allergen Ber e 2 is reported.
Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way.
brazil nut allergy; Ber e 2; cupin superfamily; allergenicity
The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 μg/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-009-2720-1) contains supplementary material, which is available to authorized users.
Biosensor; Immunoassay; Monoclonal antibody; Olive oil; Hazelnut oil; Hidden allergens
The bifunctional enzyme catalase-phenol oxidase from S. thermophilum was crystallized by the hanging-drop vapour-diffusion method in space group P21 and diffraction data were collected to 2.8 Å resolution.
Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit.
Scytalidium thermophilum; Humicola insolens; catalases; phenol oxidases; catechol oxidases; CATPO
The crystallization of peanut allergen Ara h 3 is reported.
The peanut is a significant food source, but is responsible for many cases of anaphylaxis. The peanut 11S legumin-like seed storage protein Ara h 3 is one of the best characterized allergens. In this study, Ara h 3 was extracted from peanut kernels and purified by sequential anion-exchange, hydrophobic interaction and gel-filtration chromatography to very high purity to facilitate crystallization and structural studies. Well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained and refinement of the structure is currently under way.
food safety; allergy; cupin; glycinin
A randomly sampled, cross-sectional serology test-based survey was conducted in Lebanon to describe the pattern of food allergy among Lebanese population. The prevalence of specific Immunoglobulin E (IgE) to food allergens was investigated in 20 laboratories in different regions of Lebanon by an immunoblot assay over a 1 year period. Clinical correlation was determined in two university hospitals. There were 1842 patients with suspected IgE-mediated food allergic reactions tested for specific IgE upon their physician's request. Clinical correlation was done in 93 patients. We identified 386 out of 1842 (20.95%) patients with positive specific IgE to food allergens. The clinical presentations were cutaneous, digestive, and anaphylaxis. The major cause of allergy was cow's milk in infants and young children, hazelnut and wheat flour in adults. Although specific IgE to peanut in infants, children, and adults were higher than for sesame, peanut-induced allergic reactions were mild, in contrary to sesame where anaphylaxis was the only clinical manifestation. Recently, sesame has been recognized as an increasingly frequent and potentially severe allergen. Further studies with double-blind, placebo-controlled food challenge are needed to establish the real prevalence of food allergy in Lebanon, and to determine the most common allergens taking in consideration the nutritional habits of our population.
food allergy; sesame; peanut
In this study, the core region of Ara h 1, one of the major peanut allergens, has been overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.25 Å resolution.
Peanuts contain some of the most potent food allergens known to date. Ara h 1 is one of the three major peanut allergens. As a first step towards three-dimensional structure elucidation, recombinant Ara h 1 core region was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained using 0.1 M sodium citrate pH 5.6, 0.1 M NaCl, 15% PEG 400 as precipitant. The crystals diffracted to 2.25 Å resolution using synchrotron radiation and belonged to the monoclinic space group C2, with unit-cell parameters a = 156.521, b = 88.991, c = 158.971 Å, β = 107.144°. Data were collected at the BL-38B1 station of SPring-8 (Hyogo, Japan).
peanut allergens; Ara h 1
Even 70% patients allergic to pollens of plants are developing undesirable symptoms after eating foods of the plant origin. It is most often a result of the cross-allergy between these allergens. The aim of the study was to compare the group of patients with pollinosis with patients with pollinosis and food allergy.
Fifty eight patients at the age above 16 were included in the study. Patients were divided into 2 groups. Patients included in the first group were birch allergic without any symptoms after eating food (23 persons). Patients in the other group had birch pollen allergy and they had reported clinical symptoms after eating foods such as: apple, celery, carrot, tomato, banana, peach, peanut and hazelnut (35 persons). The skin prick tests with pollen and food allergens (commercial and native) and serum IgE concentration (total and specific) were determined for all individuals. The immunoblotting was performed for the patients with the positive value of birch, apple, celery and/or carrot specific IgE to confirm the cross-reactivity.
Patients with pollinosis and symptoms after eating plant foods were characterized by a significantly larger percentage of positive skin tests with the hazel allergen. In the first group patients revealed positive results of skin tests with food allergens, although they didn't reported the problem after consumption of them. No difference in total IgE levels was found between the 2 groups (271.5 ± 403.8 IU/mL vs 242.5 ± 340.9 IU/mL). Patients with birch allergy and hypersensitivity to food allergens showed significantly higher birch pollen specific IgE levels (11.8 ± 14.1 IU/mL vs 4.1 ± 6.6 IU/mL).
Sixty percent of all the patients with birch pollinosis reported manifestations symptoms after eating certain kind of food. These patients had most often clinical symptoms after eating apples, hazelnuts and of peaches, and less frequently symptoms after eating carrots, celery, peanuts, tomatoes and bananas. Although it seems that false positive results of skin tests with food allergens in the control group and the high level of the birch specific IgE might be the predictive factor of the allergy which may develop later; they require further studies.
The Norwegian Food Allergy Register was established at the Norwegian Institute of Public Health in 2000. The purpose of the register is to gain information about severe allergic reactions to food in Norway and to survey food products in relation to allergen labelling and contamination. Cases are reported on a voluntary basis by first line doctors, and submitted together with a serum sample for specific IgE analysis. The register has received a total of 877 reports from 1 July, 2000 to 31 December, 2010. Two age groups, small children and young adults are over-represented, and the overall gender distribution is 40:60 males-females. The legumes lupine and fenugreek have been identified as two “new” allergens in processed foods and cases of contamination and faults in production of processed foods have been revealed. The highest frequency of food specific IgE is to hazelnuts and peanuts, with a marked increase in reactions to hazelnuts during the last three years. The Food Allergy Register has improved our knowledge about causes and severity of food allergic reactions in Norway. The results show the usefulness of population based national food allergy registers in providing information for health authorities and to secure safe food for individuals with food allergies.
severe reactions to food; IgE-mediated reactions to food; food allergy register; food allergens
Full-length and soluble domains of the integral membrane protein CorA have been expressed, purified and crystallized. X-ray diffraction data have been collected and analyzed.
The full-length integral membrane protein CorA from Thermotoga maritima (TmCorA1–351) has been expressed in Escherichia coli and purified without membrane isolation. TmCorA1–351 crystallized in the monoclinic space group C2, with unit-cell parameters a = 214.25, b = 86.30, c = 181.53 Å, β = 112.23°. Native crystals diffracted to 3.7 Å using synchrotron radiation, but selenomethionine-substituted crystals rarely diffracted to better than 5.0 Å. All full-length protein crystals were highly mosaic and produced anisotropic diffraction patterns. To aid in crystallographic phasing, soluble domain constructs were screened and the periplasmic domain of CorA from Archaeoglobus fulgidus (AfCorA1–263) was crystallized in the hexagonal space group P6122, with unit-cell parameters a = b = 101.17, c = 142.87 Å. Native and SeMet-substituted AfCorA1–263 crystals diffracted to ∼3.0 Å using synchrotron radiation.
membrane proteins; transporter; magnesium
Crystals of the M. sympodialis allergen Mala s 1 have been obtained using the hanging-drop vapour-diffusion method. A diffraction data set has been collected from native crystals to 1.35 Å resolution.
The opportunistic yeast Malassezia sympodialis can act as an allergen and elicit specific IgE- and T-cell reactivity in patients with atopic eczema. The first identified major allergen from M. sympodialis, Mala s 1, is present on the cell surface of the yeast. Recombinant Mala s 1 was expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Mala s 1 were obtained in 25% PEG 8K, 0.2 M (NH4)2SO4. Crystals belong to space group P21212, with unit-cell parameters a = 44.4, b = 163.7, c = 50.6 Å, and diffract to 1.35 Å resolution.
Mala s 1; Malassezia sympodialis; recombinant allergen; atopic eczema
The 11S globulin Sin a 2 is a marker to predict severity of symptoms in mustard allergic patients. The potential implication of Sin a 2 in cross-reactivity with tree nuts and peanut has not been investigated so far. In this work, we studied at the IgG and IgE level the involvement of the 11S globulin Sin a 2 in cross-reactivity among mustard, tree nuts and peanut.
Eleven well-characterized mustard-allergic patients sensitized to Sin a 2 were included in the study. A specific anti-Sin a 2 serum was obtained in rabbit. Skin prick tests (SPT), enzyme-linked immunosorbent assay (ELISA), immunoblotting and IgG or IgE-inhibition immunoblotting experiments using purified Sin a 2, Sin a 1, Sin a 3, mustard, almond, hazelnut, pistachio, walnut or peanut extracts were performed.
The rabbit anti-Sin a 2 serum showed high affinity and specificity to Sin a 2, which allowed us to demonstrate that Sin a 2 shares IgG epitopes with allergenic 11S globulins from tree nuts (almond, hazelnut, pistachio and walnut) but not from peanut. All the patients included in the study had positive skin prick test to tree nuts and/or peanut and we subdivided them into two different groups according to their clinical symptoms after ingestion of such allergenic sources. We showed that 11S globulins contain conserved IgE epitopes involved in cross-reactivity among mustard, tree nuts and peanut as well as species-specific IgE epitopes.
The allergenic 11S globulin Sin a 2 from mustard is involved in cross-reactivity at the IgE level with tree nuts and peanut. Although the clinical relevance of the cross-reactive IgE epitopes present in 11S globulins needs to be investigated in further detail, our results contribute to improve the diagnosis and management of mustard allergic patients sensitized to Sin a 2.
Food allergy; Mustard allergy; Tree nut allergy; Peanut allergy; Cross-reactivity; 11S globulins; Sin a 2; lgG/IgE epitopes
Fel d 1, a major allergen from cat, has been expressed, purified and crystallized. The crystals belong to space group P1 and diffract to 1 Å resolution.
The domestic cat (Felis domesticus) is one of the most important causes of allergic disease worldwide. A homologue of the major allergen Fel d 1 was created by linking the two chains that compose the protein. Fel d 1 (1+2) was expressed in Escherichia coli and subsequently purified using three chromatographic steps. Crystals of Fel d 1 (1+2) were obtained using the hanging-drop vapour-diffusion method in 22.5% PEG 3350, 0.5 M CaCl2. The crystals belong to space group P1, with unit-cell parameters a = 38.5, b = 42.9, c = 49.0 Å, α = 70.7, β = 80.5, γ = 81.5°, and diffract to 1.6 Å resolution.
Fel d 1; cat allergens
The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported.
Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P41 (or P43), with unit-cell parameters a = b = 150.7, c = 164.9 Å.
Pru du amandin; 11S legumins; cupin motif
Recombinant glycine decarboxylase from Synechocystis sp. PCC 6803 was expressed in E. coli and purified to homogeneity. Crystals were obtained that diffracted to 2.1 Å resolution using synchrotron radiation.
Glycine decarboxylase, or P-protein, is a major enzyme that is involved in the C1 metabolism of all organisms and in the photorespiratory pathway of plants and cyanobacteria. The protein from Synechocystis sp. PCC 6803 is a homodimer with a mass of 215 kDa. Recombinant glycine decarboxylase was expressed in Escherichia coli and purified by metal-affinity, ion-exchange and gel-filtration chromatography. Crystals of P-protein that diffracted to a resolution of 2.1 Å were obtained using the hanging-drop vapour-diffusion method at 291 K. X-ray diffraction data were collected from cryocooled crystals using synchrotron radiation. The crystals belonged to space group P212121, with unit-cell parameters a = 96.30, b = 135.81, c = 179.08 Å.
glycine decarboxylases; P-proteins
PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.
PNA; olive oil; hazelnut oil; SNP; cultivar identification; DNA fingerprinting
Different forms of the magnesium-ion transporter CorC have been crystallized; crystallization was affected by nucleotides and magnesium ions.
CorC is a magnesium transporter that is involved in the Mg2+-efflux function of the CorA transporter system, an Mg2+ channel, from Shigella flexneri. Native CorC was purified and crystallized in the native form and in a ligand-free form and diffraction data sets were collected to 2.9 and 3.4 Å resolution, respectively. The native CorC crystals belonged to space group P212121, with unit-cell parameters a = 64.31, b = 74.44, c = 132.78 Å. The ligand-free CorC crystals belonged to space group P3121/P3221, with unit-cell parameters a = b = 71.89, c = 125.96 Å. The CorC–ATP complex has also been crystallized and the crystals belonged to space group P2 or P21.
CorC; magnesium-ion transporters; Shigella flexneri; CorA transporter system
Aim. Previous studies have shown a higher sensitization rate to hazelnut in processing workers but no relation was found between the respiratory symptoms in workplace and hazelnut sensitization. Material and Method. To evaluate the association between the hazelnut sensitization and workplace-related respiratory complaints, hazelnut processing workers had undergone a questionnaire included work-related respiratory symptoms, smoking history, pulmonary function testing, and measurement of serum IgE antibodies against hazelnut. Results. This study consisted of 88 hazelnut processing workers (79 females and 9 males), aged 14–59 years (Mean ± SD: 33.8 ± 10.5 years). The mean working duration was 38.8 ± 36.6
months (min: 1–max: 180). Specific IgE against hazelnut allergens was positive in 14 of cases (17.1%). There was no significant difference between the cases with and without specific IgE against hazelnut allergens regarding respiratory symptoms, history of allergy, smoking status and spirometric values. Conclusion. 17.1% of the hazelnut processing workers were seropositive against hazelnut. Being sensitized to hazelnut was not found to be associated with work-related respiratory symptoms in this study. Further studies are needed in hazelnut workers respiratory health to search topics other than asthma.
A lectin from C. maritima was crystallized using the vapour-diffusion method and crystals diffracted to 2.1 Å resolution. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%, refinement is in progress.
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 Å resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 Å. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.
lectins; Canavalia maritima
The major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae has been crystallized and diffracted X-rays to a resolution of 2.24 Å.
Der f 7 is a major group 7 allergen from the dust mite Dermatophagoides farinae that shows 86% sequence identity to the homologous allergen Der p 7 from D. pteronyssinus. Der f 7 was successfully overexpressed in an Escherichia coli expression system and purified to homogeneity using Ni–NTA affinity and size-exclusion column chromatography. SeMet-labelled Der f 7 was crystallized by the hanging-drop vapour-diffusion method using a reservoir solution consisting of 0.1 M bis-tris pH 7.4 and 28% polyethylene glycol monomethyl ether 2000 at 293 K. X-ray diffraction data were collected to 2.24 Å resolution using synchrotron radiation. The crystals belonged to the orthorhombic system, space group P212121, with unit-cell parameters a = 50.19, b = 58.67, c = 123.81 Å. Based on the estimated Matthews coefficient (2.16 Å3 Da−1), two molecules of Der f 7 could be present in the asymmetric unit of the crystal lattice.
group 7 allergens; Der f 7; Dermatophagoides farinae
Incidence of Xanthomonas arboricola pv. corylina, the causal agent of hazelnut bacterial blight, was analyzed spatially in relation to the pedoclimatic factors. Hazelnut grown in twelve municipalities situated in the province of Viterbo, central Italy was studied. A consistent number of bacterial isolates were obtained from the infected tissues of hazelnut collected in three years (2010–2012). The isolates, characterized by phenotypic tests, did not show any difference among them. Spatial patterns of pedoclimatic data, analyzed by geostatistics showed a strong positive correlation of disease incidence with higher values of rainfall, thermal shock and soil nitrogen; a weak positive correlation with soil aluminium content and a strong negative correlation with the values of Mg/K ratio. No correlation of the disease incidence was found with soil pH. Disease incidence ranged from very low (<1%) to very high (almost 75%) across the orchards. Young plants (4-year old) were the most affected by the disease confirming a weak negative correlation of the disease incidence with plant age. Plant cultivars did not show any difference in susceptibility to the pathogen. Possible role of climate change on the epidemiology of the disease is discussed. Improved management practices are recommended for effective control of the disease.
The expression, purification and crystallization of the collagen-binding region of the E. rhusiopathiae surface protein RspB is described. The crystals diffracted to 2.2 Å resolution using synchrotron radiation.
RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB(31–348), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 Å using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 Å using synchrotron radiation. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 Å, β = 94.11°.
RspB; Erysipelothrix rhusiopathiae; collagen binding
Human S100A13 protein was cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals obtained belonged to space group P212121 and diffracted to a resolution of 1.8 Å.
S100A13 is a member of the S100 family of EF-hand-containing calcium-binding proteins and plays an important role in the secretion of fibroblast growth factor-1 and interleukin 1α, two pro-angiogenic factors released by the endoplasmic reticulum/Golgi-independent non-classical secretory pathway. Human S100A13 was heterologously expressed in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals diffracted X-rays from a synchrotron-radiation source to 1.8 Å resolution and the space group was assigned as primitive orthorhombic P212121.
S100A13; EF-hand calcium-binding proteins