Purification and Recombinant Expression of Major Peanut Allergen Ara h 1

Reaction to peanut, as one of the major food allergens, has become an increasingly common life-threatening disorder. Although peanut allergens have been extensively identified, Ara h 1 is still too expensive to be applied in food safety or clinical utility. In this study, the purification, expression, and immunological analyses of Ara h 1 are investigated. It was shown that a high purity (>95%) of Ara h 1 could be prepared by either purification or expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and mass spectroscopy were used to identify the Ara h 1, and it was found that natural Ara h 1 (nAra h 1) and expressed Ara h 1 (rAra h 1) have the same properties, including amino acid sequence. In particular, rAra h 1 reacted positively with anti-nAra h 1 serum, showing their similar immunological property. Thus, by either purification or expression, Ara h 1 could be prepared with low cost, as performed in the present work. SDS-PAGE, mag trix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), and immunological analysis confirmed that both forms of Ara h 1 had the same properties.     

Purification of swine kidney alkaline phosphatase by immunoaffinity chromatography

A homogeneous alkaline phosphatase preparation was obtained from swine kidney cortex by a simple purification step of immunoaffinity chromatography. The enzyme was purified 426 times that of the initial acetone powder with a recovery of 69.6% and a specific activity of 1206 units/mg of protein. The sodium dodecyl sulfate-gel electrophoretic pattern showed a single 80,000-M_r protein band as the monomer of the purified enzyme.     

花生主要变应原Ara h2的克隆及原核表达

目的:在原核载体中克隆、表达花生主要变应原Ara h2,为其重组变应原应用研究奠定基础。方法:合成花生主要变应原Ara h2基因,设计特异性引物行PCR扩增,经Eco RⅠ、HindⅢ双酶切后与做相应酶切的pET-28a载体连接,转化大肠杆菌BL21,提取质粒进行双酶切鉴定及测序分析,使用IPTG诱导融合蛋白表达,使用Ara h2特异性多克隆抗体对表达产物进行免疫印迹鉴定。结果:成功扩增了Ara h2基因,重组质粒双酶切见目的条带,基因测序显示Ara h2在正确开放阅读框中,基因长423bp,编码140个氨基酸,预测的等电点为5.3,分子量约16660.17 Da,基因比对分析显示其与相关报道的核苷酸序列一致性达100%。重组pET-28a-Ara h2/BL21经0.6 mmol/L IPTG诱导表达可见重组融合蛋白在相应分子量大量表达,使用Ara h2多克隆抗体免疫印迹法能检测到目的蛋白。结论:成功克隆、表达了花生主要变应原Ara h2,为其重组变应原应用研究奠定了基础。     

经合理的序列重组制备花生主要过敏原Ara h2低致敏衍生物

目的:构建经序列重组的Ara h 2表达载体,表达并纯化该蛋白,鉴定其低致敏原性.方法:根据已鉴定的Ara h 2 IgE抗原表位,利用基因工程技术将Ara h 2基因进行合理的组合,并将其序列进行合成,再将合成后的基因连入原核表达载体pET-32a(+)上,然后转入Origami宿主表达菌中;IPTG诱导表达;通过N...     

Identification and characterization of a hypoallergenic ortholog of Ara h 2.01

Peanut (Arachis hypogaea L.), can elicit type I allergy becoming the most common cause of fatal food-induced anaphylactic reactions. Strict avoidance is the only effective means of dealing with this allergy. Ara h 2, a peanut seed storage protein, has been identified as the most potent peanut allergen and is recognized by approximately 90% of peanut hypersensitive individuals in the US. Because peanut has limited genetic variation, wild relatives are a good source of genetic diversity. After screening 30 Arachis duranensis accessions by EcoTILLing, we characterized five different missense mutations in ara d 2.01. None of these polymorphisms induced major conformational modifications. Nevertheless, a polymorphism in the immunodominant epitope #7 (S73T) showed a 56–99% reduction in IgE-binding activity and did not affect T cell epitopes, which must be retained for effective immunotherapy. The identification of natural hypoallergenic isoforms positively contributes to future immunological and therapeutic studies and peanut cultivar development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.     

Rapid separation of solasodine glycosides by an immunoaffinity column using anti-solamargine monoclonal antibody

Immunoaffinity column using anti-solamargine monoclonal antibody for separation of solasodine glycosides was established. This method was specific for solasodine glycosides which was detected by thin layer chromatography and the western blotting. Total solasodine glycosides have been separated directly from the crude extract of Solanum khasianum fruit by the newly established immunoaffinity column. This revised version was published online in August 2006 with corrections to the Cover Date.     

Assessment of peanut allergen Ara h1 in processed foods using a SWCNTs-based nanobiosensor

The goals of this research were to develop a rapid single-walled carbon nanotube (SWCNT)-based biosensor and to employ it to commercial food products for Ara h1 detection. The SWCNT-based biosensor was fabricated with SWCNTs immobilized with antibody (pAb) through hybridization of 1-pyrenebutanoic acid succinimidyl ester (1-PBASE) as a linker. The resistance difference (ΔR) was calculated by measuring linear sweep voltammetry (LSV) using a potentiostat. Resistance values increased as the concentration of Ara h1 increased over the range of 1 to 10⁵ ng/L. The specific binding of anti-Ara h1 pAb to antigen including Ara h1 was confirmed by both indirect ELISA kit and biosensor assay. The biosensor was exposed to extracts prepared from commercial processed food containing peanuts, or no peanuts, and could successfully distinguish the peanut containing foods. In addition, the application of present biosensor approach documented the precise detection of Ara h1 concentrations in commercially available peanut containing foods.     

舟山眼镜蛇毒神经生长因子的亲和层析分离

用层析和制备SDS-PAGE法纯化舟山眼镜蛇(Najanajaatra Cantor)毒神经生长因子(NGF),免疫家兔获得抗血清。用辛酸-硫酸铵沉淀法初步纯化IgG,蛋白A-Sepharose亲和层析进一步纯化IgG,并与CNBr活化的Sepharose4B偶联,采用亲和层析法对舟山眼镜蛇毒神经生长因子进行分离纯化。产物经过SDS-PAGE检测呈一条带,并显示了良好的生物学活性。纯化NGF最大比活性为5.0×104U/mg蛋白,亲和常数为4.35×108L/mol,亲和层析分离NGF得率比传统分离方法得率提高35.2%,该方法为NGF的大量提取提供了技术支持。     

Alleviating peanut allergy using genetic engineering: the silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity

Peanut allergy is one of the most life-threatening food allergies and one of the serious challenges facing the peanut and food industries. Current proposed solutions focus primarily on ways to alter the immune system of patients allergic to peanut. However, with the advent of genetic engineering novel strategies can be proposed to solve the problem of peanut allergy from the source. The objectives of this study were to eliminate the immunodominant Ara h 2 protein from transgenic peanut using RNA interference (RNAi), and to evaluate the allergenicity of resulting transgenic peanut seeds. A 265-bp-long PCR product was generated from the coding region of Ara h 2 genomic DNA, and cloned as inverted repeats in pHANNIBAL, an RNAi-inducing plant transformation vector. The Ara h 2-specific RNAi transformation cassette was subcloned into a binary pART27 vector to construct plasmid pDK28. Transgenic peanuts were produced by infecting peanut hypocotyl explants with Agrobacterium tumefaciens EHA 105 harbouring the pDK28 construct. A total of 59 kanamycin-resistant peanut plants were regenerated with phenotype and growth rates comparable to wild type. PCR and Southern analyses revealed that 44% of plants stably integrated the transgene. Sandwich ELISA performed using Ara h 2-mAbs revealed a significant ( P <  0.05) reduction in Ara h 2 content in several transgenic seeds. Western immunobloting performed with Ara h 2-mAb corroborated the results obtained with ELISA and showed absence of the Ara h 2 protein from crude extracts of several transgenic seeds of the T₀ plants. The allergenicity of transgenic peanut seeds expressed as IgE binding capacity was evaluated by ELISA using sera of patients allergic to peanut. The data showed a significant decrease in the IgE binding capacity of selected transgenic seeds compared to wild type, hence, demonstrating the feasibility of alleviating peanut allergy using the RNAi technology.     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

Efficiency of a smooth desorption procedure for the purification of barley beta-amylase using immunoaffinity chromatography

Immunoaffinity chromatography was used for a one step purification procedure of beta-amylase from the G25 Sephadex gel filtrated fraction of whole barley protein extracts. The immunoglobulin G (IgG) fraction of an anti-barley beta-amylase immune serum was immobilized on Ultrogel. A gentle desorption procedure was used, combining distilled water elution with an interrupted elution. The quality of the purification was assayed by using cross immunoelectrophoresis with a polyspecific anti-barley protein immune serum. The extent of the damaging effect of this procedure was evaluated on the specific activity of the enzyme and on its polymorphism, as displayed by isoelectric focusing. The results underline the efficiency of the purification procedure and its low denaturing effect on the beta-amylase. This opens new possibilities for some aspects of the enzyme study and for the purification of other biologically active proteins.     

Development and characterization of a chitosan-supported immunoaffinity chromatography column for the selective extraction of methandrostenolone from food and feed samples

The development of a chitosan-supported immunoaffinity chromatography (IAC) column and its application to the selective extraction of methandrostenolone (MA) from food and feed samples were described in this paper. Using hybridoma technique, a monoclonal antibody (mAb) against MA was produced. The IAC column was prepared by coupling the produced antibody with crosslinked chitosan. Scanning electron microscopy and IR spectroscopy was used to characterize the chitosan crosslinking and antibody coupling. 2% and 90% methanol were respectively selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was 1790 ng/mL gel. The extraction recoveries of the column for MA at three different spiked concentrations ranged from 83.7 to 98.5%. After 2 cycles of usage, the column capacity and extraction recovery still remained 84.6% and 80.5%. To further verify the effect of matrix on the IAC cleanup, MA-fortified food and feed samples were extracted using the prepared IAC column, and MA recovery rates were found to be 86.2% and 70.4%, respectively.     

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.     

Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.     

Purification of human liver fumarylacetoacetase using immunoaffinity chromatography

A method is described to purify fumarylacetoacetase from crude human liver extracts using immunoaffinity chromatography. Immobilized rabbit antibodies specific for beef liver fumarylacetoacetase were used as an immunoadsorbent. With this rapid and specific procedure human liver fumarylacetoacetase could be purified to apparent homogeneity. The molecular weight of native human liver fumarylacetoacetase is approximately 83000 as estimated by gel filtration. The two subunits have a molecular weight of approximately 41000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Purified human liver fumarylacetoacetase has a broad pH optimum with a maximum at pH 7.2 and a K_m = 2.1 μM towards fumarylacetoacetate.     

Purification of the thermostable direct hemolysin of Vibrio parahaemolyticus by immunoaffinity column chromatography

A simple method involving immunoaffinity column chromatography to purify the thermostable direct hemolysin of Vibrio parahaemolyticus was developed. The thermostable direct hemolysin purified from the culture supernatant of a strain isolated from the first reported case of V. parahaemolyticus infection in China in 1985 was indistinguishable from the hemolysins purified from strains isolated in Japan.     

Efficient recovery of recombinant proteins using membrane-based immunoaffinity chromatography (MIC)

A systematic approach to the design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon-alpha2a is used as a model system to determine the operational parameters in membrane-based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen-antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, have been purified efficiently employing membrane-based immunoaffinity chromatography. Overall, membrane-based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial-scale production of recombinant proteins. (c) 1992 John Wiley & Sons, Inc.     

Purification of a brain-specific astroglial protein by immunoaffinity chromatography

An immunoaffinity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglogulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immuno-affinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 m acetic acid, 5 m urea, 0.8 m sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mm to 1 mm sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.     

Purification of an enterotoxin produced by Bacillus cereus by immunoaffinity chromatography using a monoclonal antibody.

A murine monoclonal antibody (MAb) with high reactivity to an enterotoxin produced by Bacillus cereus was used to prepare an immunoadsorbent for purification of the enterotoxin. By immunoaffinity chromatography using the immunoadsorbent, approximately 25% of crude enterotoxin applied was recovered in the eluate. The purified enterotoxin was found to be electrophoretically and antigenically homogeneous. It also showed vascular permeability activity and mouse lethality, and caused fluid accumulation in mouse ligated intestinal loops, whereas it did not show any hemolytic and lecithinase activities. Thus, immunoaffinity chromatography proved useful in the purification of enterotoxin produced by B. cereus in terms of recovery, purity, and relative ease of performing the purification.