2014 Volume 20 Issue 4 Pages 875-881
Simplified methods to purify major peanut allergens (Ara h 1, Ara h 2, and Ara h 3) were investigated to facilitate current laborious and time-consuming conventional purification methods, the combination of ammonium sulfate fractionation (ASF) and successive column chromatographic separations. Crude peanut proteins obtained from defatted peanut were subjected to ASF at higher concentrations of ammonium sulfate than those in conventional methods and then dialyzed against water. Purified Ara h 1, Ara h 2, and Ara h 3 were obtained by ultrafiltration(s) of the dialyzed supernatant in ASF (80 – 90%), the dialyzed precipitate in ASF (40 – 50%), and the dialyzed precipitate in ASF (0 – 40%), respectively. The purities of Ara h 1, Ara h 2, and Ara h 3 were 91.3%, 86.4%, and 93.9%, respectively. The simplified methods enable the purification of the three major peanut allergens in a relatively short time without the use of chromatographic separation techniques.
The recent increased prevalence of food allergy has become a serious issue primarily in developed countries. For instance, 3.5% of the total population in France (Kanny et al., 2001), 3.6% in Germany (Zuberbier et al., 2004), and 3.5 – 4.0% in the United States (Muñoz-Furlong et al., 2004) have experienced food allergy. In Japan, the prevalence of food allergy is approximately 5 – 10% in infants, 2% in school children (Uris et al., 2011), and 1 – 2% in the total population (Ebisawa, 2009).
Among allergenic foods, peanut can induce a fatal systemic anaphylaxis reaction; thus, special attention should be paid to peanut. In addition, peanut allergy tends to persist for life, while tolerances to other allergenic foods such as egg, milk, and wheat are generally acquired with aging. Therefore, peanut allergy is a disease that has significant adverse effects on the quality of life (QOL) of patients and their families.
Notably, a large number of peanut allergy patients are found in North America and Europe, where a large amount of peanut is consumed. In the United States, more than 1% of the population is allergic to peanut and peanut allergy is the leading cause of fatal food-induced allergic reactions (Sicherer et al., 2010). The prevalence of peanut allergy is showing an increasing tendency in Japan. Nationwide monitoring investigations on food allergy in Japan revealed that the ratio of peanut allergy patients to total patients suffering from food allergy was 2.8% in 2002 (Ebisawa and Imai, 2004), 4.2% in 2005 (Ebisawa and Imai, 2005), and 4.8% in 2008 (Imai et al., 2008).
In peanut, 11 allergenic proteins have been reported, and Ara h 1, Ara h 2, and Ara h 3 are known as the major allergens (Burks et al., 1998).
Ara h 1 was the first protein identified as a peanut allergen; it is a seed storage protein classified as a 7S globulin. Ara h 1 is the most abundant of the peanut seed proteins, accounting for 12 – 16% of the total protein content in peanut (Koppelman et al., 2001). It is utilized as a nitrogen source during peanut seedling growth (Maleki et al., 2000). Ara h 1 is recognized by more than 90% of serum IgE from peanut allergic patients, indicating its importance in the etiology of peanut allergy (Burks et al., 1995). Ara h 1 exists as a stable trimer of monomers (∼ 64 kDa) bound to each other (Shin et al., 1998). The stable structure of the trimer contributes to its resistance to protease digestion (van Boxtel et al., 2008).
Ara h 2 is a seed storage protein classified as a 2S albumin, and accounts for 5.9 – 9.3% of the total protein content in peanut (Koppelman et al., 2001). It comprises two isoforms (∼ 16 kDa and ∼ 18 kDa); the larger contains a 12 amino acids insertion, which accounts for the difference in molecular mass (Chatel et al., 2003). As with Ara h 1, Ara h 2 is recognized by more than 90% of serum IgE from peanut allergic patients (Stanley et al., 1997). It forms a stable structure containing four disulfide bonds in the molecule, and its resistance to protease digestion is higher than Ara h 1 and Ara h 3 (Koppelman et al., 2010). In addition, the ability of Ara h 2 to release histamine from human basophils and its skin test reactivity are much higher than those of Ara h 1 and Ara h 3, respectively. Therefore, this is a strong evidence that Ara h 2 is the most important of the peanut allergens (Koppelman et al., 2004).
Ara h 3 is classified into the 11S globulins as a seed storage protein and is recognized by more than 50% of serum IgE from peanut allergy patients (Kleber et al., 1999). The single chain protein (∼ 58 kDa) is cut into 13 – 38 kDa acidic subunits and a 21 kDa basic subunit by protein processing, and the processed subunits are linked by disulfide bonds (Piersma et al., 2005). The bound subunits as monomers form a hexamer (Jin et al., 2009).
In studies on peanut allergens, such as the elucidation of structures and properties, as well as the reduction of immunoreactivities, it is indispensable to purify target peanut allergenic proteins. In previous reports, target allergens have been purified as follows: protein extraction from defatted peanut by using a weakly basic buffer solution, ammonium sulfate fractionation (ASF) once or twice, and final purification steps using a combination of several column chromatography techniques, such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography (Maleki et al., 2000; van Boxtel et al., 2006; Burks et al., 1992; de Jong et al., 1998; Sen et al., 2002; Koppelman et al., 2003).
As described above, column chromatography has been an indispensable method for the purification of peanut allergens in conventional protocols. Although a powerful tool for obtaining target proteins of high purity, a number of preparative procedures are required, such as column setting, column equilibration with initial eluent, exchanging sample matrix, and adjusting sample concentration, as well as successive procedures for elution and fraction collection. In addition, after collection of fractions, concentration (e.g., ultrafiltration), desalting (e.g., dialysis), and exchanging the solution matrix are often required. Thus, the procedures for column chromatography are time-consuming and laborious; for instance, the conventional purification of peanut allergens using column chromatography requires at least 3 days: a preparative procedure (1 day) and successive ones (2 days). Furthermore, prior to the column chromatography process, a pre-treatment process of at least 3 days is necessary, which involves preparation of defatted peanut powder, protein extraction, ASF, and dialysis.
In this study, to minimize the time requirement and complicated processes of peanut allergen purification, simplified methods were investigated by combining ASF, dialysis, and ultrafiltration. The aim was to efficiently obtain sufficient amounts of peanut allergens for use in fundamental studies of the consequence of physico-chemical treatments on peanut allergenicity.
Preparation of defatted peanut powder Peanut seeds (Arachis hypogaea L., cv. Shaobaisha) harvested in 2009 in Liaoning province of China were used in this study. Peanut seeds were peeled and ground by a laboratory scale mill (Millcer, Iwatani Co. Ltd., Japan). Diethyl ether was added to the ground peanut and the mixture was stirred thoroughly. After centrifugation of the mixture, the supernatant was discarded and the precipitate was suspended in diethyl ether and stirred thoroughly. After centrifugation, the supernatant was discarded and the precipitate was air-dried in a draft chamber overnight to evaporate the diethyl ether. The dried powder was used as defatted peanut.
Extraction of crude peanut protein To 1 g of defatted peanut powder, 20 mL of Tris-HCl buffer solution (TB; 50 mM Tris, 200 mM NaCl, pH 8.3) was added and the mixture was stirred at 4°C for 1 h. After centrifugation, the supernatant was filtrated with qualitative filter paper (No. 2, Advantec, Japan) and a crude protein extract was obtained.
Ammonium sulfate fractionation (ASF) In conventional methods, the crude protein extract is fractionated using ammonium sulfate (AS) precipitation at 0 – 40%, 40 – 70%, and 70 – 100% saturations. In this study, fractionation was carried out using ammonium sulfate precipitation at 0 – 40%, 40 – 50%, 50 – 60%, 60 – 70%, 70 – 75%, 75 – 80%, and 80 – 90% saturations. Each precipitate obtained by ASF was dissolved in TB, centrifuged at 2500 × g for 10 min at 4°C to remove insoluble particles, and dialyzed against distilled water for a minimum of one night. The insoluble fraction during dialysis was separated from the soluble supernatant by centrifugation. Soluble and insoluble fractions were lyophilized and solubilized in TB for successive ultrafiltration step(s).
Ultrafiltration Following the extraction and ASF, ultrafiltration was carried out once for Ara h 1 and Ara h 3 and twice for Ara h 2. A syringe-pressurizing type ultrafiltration unit (USY-20; 200,000 molecular weight cut off (MWCO); Advantec, Japan) was used for the purification of Ara h 1 and Ara h 3. Centrifugal ultrafiltration units (VIVASPIN 20; 30,000 and 3,000 MWCO; Sartorius Stedim, Germany) were used in the ultrafiltration steps for Ara h 2: the filtrate from ultrafiltration using the 30,000 unit was then concentrated and recovered using the 3,000 unit.
Protein quantification Protein in solution was quantified by the Lowry method using a protein assay kit (DC Protein Assay Kit; Bio-Rad, USA) with bovine serum albumin (Sigma Aldrich, St. Louis, MO, USA) as a standard.
Electrophoresis Electrophoresis was performed based on the method of Laemmli (Laemmli, 1970) using the MiniProtean System (Bio-Rad, USA) and prepared TGX Gel (4 – 20% gradient polyacrylamide gel; Bio-Rad, USA). Laemmli sample buffer solution (62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 2% SDS, and 0.01% bromophenol blue; Bio-Rad, USA) containing 5% β-mercaptoethanol was added to an equal volume of protein sample solution. The mixture was electrophoresed at 200 V. After electrophoresis, the gel was stained with Bio-safe Coomassie Stain (Bio-Rad, USA). The stained gel was scanned using an image scanner connected to a personal computer, and the intensity of the bands was analyzed using Image J software (Schneider et al., 2012).
N-terminal amino acid sequence To identify each obtained protein, N-terminal amino acid sequence (8 – 10 residues) was determined by the Edman degradation method. With Ara h 1 and Ara h 2, the protein samples after ultrafiltration(s) were analyzed. With Ara h 3, the protein sample after ultrafiltration was electrophoresed, and the protein bands (37 kDa and 21 kDa) that were electroeluted and collected from the SDS-PAGE gel using an electroeluter (Mini Whole Gel Eluter, Bio-Rad) were analyzed.
Purification of Ara h 1 In conventional procedures, column chromatography is applied for the purification of Ara h 1 as described above. For instance, Ara h 1 is purified as follows: the peanut protein extract is subjected to ammonium sulfate fractionation (ASF), and after removal of the precipitate fraction at 0 – 70% AS, the precipitate fraction at 70 – 100% AS is collected. The collected fraction is then purified by cation exchange chromatography (Maleki et al., 2000). In another procedure, the peanut protein extract is fractionated by gel permeation chromatography twice (van Boxtel et al., 2006).
For comparison with the conventional procedure, ASF was carried out according to the conventional method. The fractionation condition employed was at 0 – 70% AS and 70 – 100% AS saturation (Maleki et al., 2000). In the 70 – 100% AS fraction, Ara h 1 was recovered at ∼ 64 kDa, as well as proteins at 11 – 15 kDa, ∼ 21 kDa, and ∼ 37 kDa (Fig. 1, lane 4).
SDS-PAGE patterns of ammonium sulfate fractions in Ara h 1 purification
lane 1, protein markers; lane 2, crude peanut proteins; lane 3, (ASF 0 – 70%) + Dialysis (sup); lane 4, ASF (70 – 100%) + Dialysis sup) ; lane 5, ASF (70 – 75%) + Dialysis (sup); lane 6, ASF 75 – 80%) + Dialysis (sup); lane 7, ASF (80 – 90%) + Dialysis sup); lane 8, ASF (80 – 90%) + Dialysis (sup) + UF.
ASF; ammonium sulfate fractionation, sup; supernatant, UF; ultrafiltration
The 21 kDa and 37 kDa proteins might be Ara h 3 basic and acidic subunits, respectively (Piersma et al., 2005). Native Ara h 3 is composed of subunits linked intermolecularly by disulfide bonds to form a monomer, and the monomers further form a hexamer (∼ 350 kDa) (Jin et al., 2009). Notably, contaminating fractions at ∼ 21 kDa and ∼ 37 kDa, supposedly derived from Ara h 3, were observed in the high molecular mass fraction in the successive ultrafiltration (200,000 MWCO), similar to the 64 kDa fraction (data not shown).
In this study, ASF was carried out under a more detailed condition than that of the conventional process. It was found that fractionation at 70 – 75% AS and 75 – 80% AS following 0 – 70% ASF eliminated the 37 kDa and 21 kDa contaminating proteins from the fraction at 80 – 90% AS (Fig. 1, lane 7). On the other hand, 70 – 80% ASF following 0 – 70% ASF did not eliminate the 37 kDa and 21 kDa proteins from the fraction at 80 – 90% AS (data not shown). Since the elimination of Ara h 3 is key in the purification of Ara h 1, the 70 – 75% and 75 – 80% ASFs are indispensable procedures.
The precipitate of 80 – 90% ASF was dialyzed, and the dialyzed supernatant (5 mg) was dissolved in 1 mL of TB and centrifuged at 9000 × g for 10 min at 4°C. The supernatant (0.8 mL) was applied to ultrafiltration (200,000 MWCO) so as to remove the low molecular mass fraction (around 11 – 15 kDa) observed in lane 7 in Fig. 1. The low molecular mass fraction was eliminated by repeated addition of TB (total 8 mL) and concentration, and the protein was observed as a single 64-kDa band on SDS-PAGE (Fig. 2, lanes 3 and 4). The N-terminal amino acid sequence of the single-band protein was analyzed, and the protein was confirmed to be Ara h 1.
SDS-PAGE patterns of Ara h 2 and Ara h 3 fractions purified by ASF and dialysis in combination with successive UF
lane 1, protein markers; lane 2, crude peanut proteins; lane 3, ASF (40 – 50%) + Dialysis (ppt); lane 4, ASF (40 – 50%) + Dialysis (ppt) + UF; lane 5, ASF (0 – 40%) + Dialysis (ppt); lane 6, ASF (0 – 40%) + Dialysis (ppt) + UF; ASF; ammonium sulfate fractionation, sup; supernatant, ppt; precipitate, UF; ultrafiltration.
In the ultrafiltration process, several kinds of ultrafiltration membranes were examined (data not shown). It was found that the 200,000 MWCO ultrafiltration membrane removed the low molecular mass proteins the most efficiently. Since native-state Ara h 1 exists as a trimer (∼ 190 kDa) of the monomer (∼ 64 kDa), the membrane provides a suitable condition, in terms of the MWCO, to reject and concentrate Ara h 1 and to pass and remove the low molecular mass fractions efficiently.
The degrees of purification and yields for Ara h 1 purification are listed in Table 1. The degree of purification was expressed as a ratio of allergen protein to total proteins for each fraction. The values are the averages of three different samples. The degree of purification for Ara h 1 was 91.3%.
| Purification Step | Total Protein (mg) | Degree of urification (%) | Allergen Content (mg) | Yield (%) | |
|---|---|---|---|---|---|
| Ara h 1 | Defatted Peanut | 60.3 | 33.4 | 20.1 | 100.0 |
| ASF (80 – 90%) + Dialysis (sup) | 3.1 | 59.2 | 1.8 | 9.1 | |
| Ultrafiltration (Concentrate) | 1.5 | 91.3 | 1.4 | 6.8 | |
| Ara h 2 | Defatted Peanut | 19.2 | 12.7 | 2.4 | 100.0 |
| ASF (40 – 50%) + Dialysis (ppt) | 2.9 | 33.1 | 1.0 | 39.5 | |
| Ultrafiltration (Concentrate) | 0.2 | 86.4 | 0.2 | 7.1 | |
| Ara h 3 | Defatted Peanut | 19.9 | 38.7 | 7.7 | 100.0 |
| ASF (0 – 40%) + Dialysis (ppt) | 2.8 | 85.0 | 2.4 | 30.9 | |
| Ultrafiltration (Concentrate) | 2.3 | 93.9 | 2.2 | 28.0 |
ASF, ammonium sulfate fractionation; sup, supernatant; ppt, precipitate
Degree of purification was quantified by image analysis of the band intensities in SDS-PAGE and expressed as ratios of allergen proteins to total protein of each fraction. The values are averages of three independent samples.
Purification of Ara h 2 In conventional methods for Ara h 2 purification, column chromatography has been indispensable, as in the case of Ara h 1. For instance, a peanut protein extract was fractionated by anion and cation exchange chromatography once or twice (Burks et al., 1992; de Jong et al., 1998), and the precipitate fraction of 40 – 70% ASF from the extract was further fractionated by anion exchange chromatography, followed by hydrophobic chromatography (Sen et al., 2002). On the SDS-PAGE gel, Ara h 2 can be found as two bands at ∼16 kDa and ∼18 kDa, representing 2 isoforms containing different numbers of repeated inserted sequence (Chatel et al., 2003).
In conventional methods for Ara h 2 purification, ASF as the first step was conducted at 40 – 70% AS. In this study, ASF was carried out under a more detailed AS condition, and each fraction was further dialyzed to water. It was shown that the 40 – 50% ASF precipitate was dialyzed and Ara h 2 was highly concentrated, and contamination with proteins of smaller molecular mass than Ara h 2 was minimized (Fig. 2, lane 3).
The fraction containing Ara h 2 was lyophilized, and a portion (5 mg) of the lyophilized powder was dissolved in 1 mL of TB and centrifuged at 9000 × g for 10 min at 4°C. The supernatant (0.8 mL) was subjected to ultrafiltration (30,000 MWCO) followed by repeated addition of distilled water (total 10 mL) and concentration.
The filtrate was further subjected to ultrafiltration (3,000 MWCO) and the concentrate was recovered. The proteins in the filtrate showed a typical electrophoretic pattern of Ara h 2 on SDS-PAGE (Fig. 2, lane 4). The Ara h 2 protein comprises two isoforms, and the pattern showed two bands at ∼ 16 and ∼ 18 kDa. The isoforms share an identical N-terminal amino acid sequence (Chatel et al., 2003), and the analyzed sequences confirmed that the two bands were derived from Ara h 2 isoforms, respectively. The degree of purification was 86.4% (Table 1).
In establishing the above-mentioned protocol for the purification of Ara h 2, a number of challenges were addressed in advance. One was the ultrafiltration unit for the first ultrafiltration. After dialyzing the 40 – 50% ASF precipitate, the dialysate was first ultrafiltrated using the syringe pressurizing ultrafiltration unit (50,000 MWCO). However, Ara h 2 and other 20 – 30 kDa proteins passed through the membrane and the purification of Ara h 2 failed (data not shown). On the other hand, another unit possessing a 30,000 MWCO membrane rejected the 20 – 30 kDa proteins, and proteins smaller than 20 kDa containing Ara h 2 were successfully obtained in the filtrate. Thus, the ultrafiltration unit was optimized. Another challenge involved the liquid used for dissolving the lyophilized powder in the first ultrafiltration. When TB was used for the ultrafiltration with the 30,000 unit, proteins of ∼ 23 kDa and ∼ 30 kDa, including Ara h 2, passed through the membrane unit and the purification of Ara h 2 was not successful (data not shown). However, by using water instead of TB the purification was successfully carried out as mentioned above. It was speculated that the contaminating proteins might be less solubilized in water than in TB or they might form large molecules in distilled water, preventing passage through the membrane.
Notably, an ∼ 15 kDa protein was an occasional contaminant of Ara h 2. The protein is suspected to be Ara h 6, a 2S albumin and a homologue of Ara h 2. The amino acid sequence of Ara h 6 shares 59% identity with Ara h 2, and its resistance to protease digestion and allergenic potency are comparable to those of Ara h 2 (Koppelman et al., 2010; Koppelman et al., 2005). Ara h 2 and Ara h 6 have been occasionally used without separation (Vissers et al., 2011; Kulis et al., 2011). Since the molecular mass of Ara h 6 is lower than that of Ara h 2, it can pass through the 30,000 MWCO ultrafiltration membrane. Therefore, the use of another ultrafiltration unit with a MWCO < 30,000 is suggested. Further study on the ultrafiltration process may be required to improve the purity and yield of Ara h 2.
Purification of Ara h 3 In conventional purification of Ara h 3, the peanut protein extract is fractionated by anion exchange chromatography (Koppelman et al., 2003). Structural analysis of the purified Ara h 3 by MALDI-TOF-MS revealed varied structures composed of both acidic subunits (∼ 13 – 38 kDa), which are processed at specific sites, and basic subunits (∼ 21 kDa). Furthermore, both subunits are linked by disulfide bonds in complicated manners (Piersma et al., 2005). Since various processing products of Ara h 3 migrated as multiple bands on the SDS-PAGE gel, it was difficult to distinguish Ara h 3 bands from the other contaminating proteins, making it challenging to evaluate the purity of Ara h 3. Therefore, the total band intensity of the bands at ∼ 37 kDa, ∼ 34 kDa, ∼ 25 kDa, and ∼ 21 kDa on the gel was designated as Ara h 3 and used for the purity evaluation.
The combination of ASF and dialysis revealed that the 4 peptides derived from Ara h 3 were concentrated in the dialyzed fraction of the precipitates of 0 – 40% ASF (Fig. 2, lane 5). The lyophilized fraction (5 mg) was dissolved in 1 mL of TB and centrifuged at 9000 × g for 10 min at 4°C. A portion (0.8 mL) of the supernatant was applied to the 200,000 MWCO ultrafiltration unit. Low molecular mass fractions were removed by repeated addition of TB (total 8 mL) and concentration. The target protein showed the typical electrophoretic pattern of Ara h 3 (Fig. 2, lane 6). The N-terminal amino acid sequences of the 21 kDa and 37 kDa bands were analyzed, and it was confirmed that the 2 peptides originated from Ara h 3.
Ara h 3 exists as a relatively large 350 kDa hexamer in its native state (Jin et al., 2009). Due to its large size, Ara h 3 was effectively rejected by the 200,000 MWCO ultrafiltration membrane and thus concentrated, while low molecular mass contaminating proteins were effectively removed in the filtrate.
After ASF followed by dialysis, the purity of the precipitate fraction at 0 – 40% AS was 85.0% (Table 1), indicating that most of the contaminating proteins were removed from the fraction. Ultrafiltration of the fraction removed contaminating proteins with a molecular mass of < 20 kDa, and a relatively high purity of > 90% was achieved for the purification of Ara h 3.
Advantages of the simplified purification methods In conventional purification of Ara h 1, Ara h 2, and Ara h 3, methods based on column chromatography have been adopted. Although the purities can be sufficiently high from the viewpoint of currently available purification technologies, column chromatography is both time consuming and labor intensive. Following chromatography, the sample must be subjected to further treatments such as concentration, desalting, and exchanging buffer solution.
Figure 3 shows the purification protocol for peanut allergens Ara h 1, Ara h 2, and Ara h 3 in this study. Not only do the simplified methods not require complicated procedures, purification time is shortened by 1 or 2 day(s). Moreover, ultrafiltration as a final step enabled concentration, desalting, and exchanging buffer solution at one time, streamlining the entire procedure.
Purification protocols for Ara h 1, Ara h 2, and Ara h 3 TB, Tris-HCl buffer solution (50 mM Tris, 200 mM NaCl, pH = 8.3); SUP, supernatant; PPT, precipitate; FLT, filtrate; CONC, concentrate
Another advantage of the simplified methods will be the savings realized in the cost of large-scale purification. With conventional methods, large-scale purification is possible but expensive. This is especially true for column chromatography, in which expensive equipment such as special thick and long columns, detectors, and pumps are required. In contrast, in the simplified methods of this study, purification scale-up by using large-scale ASFs and ultrafiltration units will be much easier than with the conventional methods.
It is expected that our research group will employ the purified peanut allergens in the future to investigate the consequence of physico-chemical treatments on allergenicity.
