Isolation,cloning, and characterization of the 2S albumin: A new allergen from hazelnut

Scope: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. Methods: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP‐HPLC. After N‐terminal sequencing, degenerated and poly‐d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut‐allergic patients. Results: N‐terminal sequencing of a ～10 kDa peak from size exclusion chromatography/RP‐HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N‐terminal and a 10aa internal peptide. The obtained clone (441 bp) encoded a 147aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. Conclusion: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.     

Validating allergen coding genes (Cor a 1, Cor a 8, Cor a 14) as target sequences for hazelnut detection via Real-Time PCR

Hazelnuts have been shown to contain different allergenic proteins. Amongst these, major allergens Cor a 1 and Cor a 8 and the 2S albumin Cor a 14 were selected as targets to comparatively validate three Real-Time PCR protocols. We investigated both on the choice of the amplification target, and on the matrix effect on different sample foods. Applying statistics on the validation parameters obtained from the three protocols, we showed a significant difference in Ct values. This could turn critical when a high sensitivity method is required for the detection of hazelnut traces, confirming how fundamental the choice of the template during primer design phase is. Concluding, statistical approach represents a useful tool for the identification of the best performing primer pairs in Real-Time PCR. Cor a 8 gene permitted the identification of hazelnut based ingredients in complex foods, providing a significantly higher sensitivity in the PCR amplification, when compared to Cor a 1 and Cor a 14.     

基于榛子过敏原2S albumin基因的实时荧光聚合酶链式反应检测方法开发

目的 开发一种基于2S albumin基因的实时荧光聚合酶链式反应检测方法(real-time fluorescent polymerase chain reaction,qPCR)用于检测食品中的榛子成分。方法 采用qPCR技术,对建立的方法的特异性、扩增效率、检出限和稳健性进行验证。结果 本方法对检测目标呈现出高特异性,与其余12种非目标样品无交叉反应。扩增效率为99.42%,相关系数r2为0.9941,两者均符合实时荧光PCR方法验证指南要求。该方法检出限（limit of detection,LOD6）为每个反应5拷贝（2.5 copies/μL）,可检测出食品中含有的微量榛子成分。使用正交实验设计评估该方法具有很好的稳健性（P=0.07）,可转移到其他实验室及常规分析中使用。结论 基于2S albumin基因组分的qPCR检测方法可用于识别食品中潜在的榛子成分,对于预防榛子过敏及开发减敏脱敏榛子食品研究具有很好的技术支撑。     

Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.     

Purification and characterisation of the 7S globulin storage protein from sesame (Sesamum indicum L.)

The 7S globulin from sesame seeds was purified by means of selective precipitation and anion-exchange chromatography on Q Sepharose Fast Flow. The 7S globulin migrated as a single band on native PAGE, which suggested homogeneity of the sample. The isolated protein was composed of at least eight polypeptide chains, ranging from 12.4 to 65.5 kDa, judged by SDS–PAGE analysis, and did not contain disulphide bonds. Furthermore, comparison of the polypeptide bands of the 7S and 11S globulins by SDS–PAGE indicated that the purified 7S globulin was free of legumin-like contaminant polypeptides and of 2S albumin. The identity of the purified polypeptides was verified by comparing the N-terminal amino acid sequences of the main polypeptide bands with the amino acid sequence deduced from a cDNA clone, which encoded the sesame 7S globulin precursor. Purification of the 7S globulin from sesame has not previously been reported.     

Development of a three‐tier in vitro system,using Caco‐2 cells,to assess the effects of lactate on iron uptake and transport from rye bread following in vitro digestion

A three‐tier Caco‐2 cell system was developed to assess simultaneously iron dialysability, uptake and transport across the Caco‐2 monolayer from an in vitro digested food matrix. The effect of lactate (0–200 mmol L⁻¹) on iron absorption from rye bread subjected to simulated peptic (pH 5.5) and pancreatic digestion (pH 6.5) was investigated to model absorption pre and post the sphincter of Oddi. Lactate increased dialysability (11.8%, P < 0.05) in peptic digests whereas it reduced it in pancreatic digests (4.9%, P < 0.001). Iron uptake from the peptic digests was in the region of 39–76 pmol mg⁻¹ protein whereas it decreased from 281 to 51 pmol mg⁻¹ protein in pancreatic digests. Iron transport was calculated for the peptic digests from [¹⁴C]polyethylene glycol movement and only at 200 mmol L⁻¹ lactate was there any detectable transcellular transport (180 pmol mg⁻¹ protein, P < 0.05). Iron absorption was positively correlated to dialysable iron for both digests (R² = 0.48 and 0.41, respectively, P < 0.01) and the effect of lactate was therefore associated mainly with iron bioaccessibility. The three‐tier system showed the potential to obtain detailed insight into each step involved in iron transport across the monolayer from a food mixture. Copyright © 2006 Society of Chemical Industry     

X. Yeast sequencing reports. The sequence of a 36 kb segment on the left arm of yeast chromosome X identifies 24 open reading frames including NUC1, PRP21 (SPP91), CDC6, CRY2, the gene for S24, a homologue to the aconitase gene ACO1 and two homologues to chromosome III genes

A 36 kb fragment from the left arm of chromosome X, located at about 50 kb from the telomere, was sequenced and analysed. The segment contains a new putative ARS, a new tRNA for threonine, remnants of a solo delta and 24 open reading frames (ORFs) numbered from J0310 to J0355. Six of them, NUC1, PRP21 (also called SPP91), CDC6, CRY2, the gene encoding the ribosomal protein S24 and the gene coding for a hypothetical protein of 599 amino acids, have been sequenced previously. Three ORFs show high homology to the yeast gene ACO1 encoding mitochondrial aconitase and to the chromosome III genes YCR34W and YCR37C of unknown function. Three other ORFs show lower but significant homology: a first one to UNP, a gene related to the tre-2 oncogene from mouse and to the gene coding for the yeast deubiquitinating enzyme DOA2; a second one to SLY41, a suppressor of the functional loss of YPT1 and a third one to the gene encoding the proline utilization activator PUT3. The complete nucleotide sequence of 36 016 bp was submitted to the EMBL database (accession number X77688).     

Neriifolin S, a dimeric serine protease from Euphorbia neriifolia Linn.: Purification and biochemical characterisation

A dimeric serine protease Neriifolin S of molecular mass 94 kDa with milk clotting activity has been purified from the latex of Euphorbia neriifolia by anion exchange and size-exclusion chromatography. It hydrolyses peptidyl substrates l-Ala-pNA with highest affinity (K_m of 0.195 mM) and physiological efficiency (K_cat/K_m of 144.5 mM s). Enzyme belongs to the class of neutral proteases with pI value of 6.8, optimal proteolytic activity displayed at pH 9.5 and temperature 45 °C. Its proteolytic activity is strongly stimulated in the presence of Ca⁺² ions and exclusively inhibited by serine protease inhibitors. Enzyme is fairly stable toward chemical denaturants, pH and temperature. The apparent T_m, was found to be 65 °C. Thermal inactivation follow first order kinetics with activation energy (Ea), activation enthalpy (ΔH∗), free energy change (ΔG∗) and entropy (ΔS∗) of 27.54 kJ mol⁻¹, 24.89 kJ mol⁻¹, −82.34 kJ mol⁻¹ and 337.20 J mol⁻¹ K⁻¹.