Article pubs.acs.org/JAFC
Simple, Rapid, and Selective Isolation of 2S Albumins from Allergenic Seeds and Nuts Marlene Hummel, Tina Wigger, Tessa Höper, Imke Westkamp, and Jens Brockmeyer* Institute of Food Chemistry, Westfälische Wilhelms-Universität Münster, Corrensstraße 45, 48149 Münster, Germany S Supporting Information *
ABSTRACT: The 2S albumins belong to the group of seed storage proteins present in different seeds and nuts. Due to their pronounced allergenic potential, which is often associated with severe allergic reactions, this protein family is of special interest in the field of allergen research. Here we present a simple, rapid, and selective method for the purification of 2S albumins directly from allergenic seeds and nuts. We systematically optimized the parameters “buffer system”, “extraction temperature”, “buffer molarity”, and “pH ” and were able to achieve 2S albumin purities of about 99% without further purification and demonstrate transferability of this method to nine different allergenic food matrices. Compared to conventional isolation routines, significant reduction of hands-on time and required laboratory equipment is achieved, but nonetheless higher protein yields are obtained. The presented method allows for the rapid purification of different 2S albumins including the corresponding isoforms from natural material. KEYWORDS: 2S albumins, extraction, purification, isolation, seeds, nuts, allergens, seed storage proteins, SDS-PAGE
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INTRODUCTION The 2S albumins belong to the superfamily of prolamins and form a group of allergenic seed storage proteins present in numerous dicotyledonous plant species.1 They are localized in the protein bodies formed during seed maturation and provide a store for amino acids utilized during germination and seedling growth.2,3 In addition to this plant physiological role, 2S albumins from several species were described as major allergens, namely, 2S albumins from mustard, sesame, cashew nut, Brazil nut, and others,3 indicating that this protein family is a panallergen. The 2S albumins are heterodimeric proteins synthesized from a single ∼13 kDa precursor protein that is cleaved into a small subunit (3−4 kDa) and a large subunit (9−10 kDa) during posttranslational processing.3,1 Circular dichroism (CD) studies demonstrate that 2S albumins from different plant species (including mustard) mainly consist of α-helical secondary structure elements (35−50%).3,4 The isolation of 2S albumins is highly relevant for different scientific applications in the field of allergen research. One of these applications is the comprehensive analysis of 2S albumin heterogeneity. We recently performed the characterization of the 2S albumin Sin a 1 from mustard seeds,5 and Downs et al. investigated the low molecular weight allergens (including the 2S albumin Jug r 1) in a similar study.6 In addition, selective extraction of single allergens from natural sources with high yield might form the basis for component-resolved diagnosis (CRD) that takes into account natural variants and isoforms of the respective allergen. Several methods were developed for the purification of 2S albumins, including ion exchange chromatography, size exclusion chromatography (SEC), and reversed-phase highperformance liquid chromatography.7−10 Approaches to directly isolate 2S albumins from plant material in general resulted in limited purity of extracts.11 Here, we present the © XXXX American Chemical Society
comprehensive optimization of parameters affecting the selectivity and yield of 2S extraction and present to the best of our knowledge the first generally applicable method for the selective extraction of these allergens with high yield from different plant materials.
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MATERIALS AND METHODS
Materials and Reagents. White mustard (Sinapis alba (L.)) seeds of four different brands were purchased from local supermarkets and blended to exclude cultivar-specific effects. Common sunflower (Helianthus annuus (L.)) seeds, common hazel (Corylus avellana (L.)) nuts, pistachio (Pistacia vera (L.)) seeds, Brazil nut (Bertholletia excelsa (L.)) seeds, buckwheat (Fagopyrum esculentum (L.)) seeds, chickpea (Cicer arietinum (L.)) seeds, sesame (Sesamum indicum (L.)) seeds, pecan (Carya illinoinensis (L.)) seeds, cashew (Anacardium occidentale (L.)) nut seeds, walnuts (Juglans regia (L.)), peanut (Arachis hypogaea (L.)) seeds, and soybean (Glycine max (L.)) seeds were purchased from local supermarkets. All nuts and seeds were stored at −20 ± 2 °C and were used within 3 months. Glycine, sodium acetate, sodium citrate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (Tris), sodium hydrogen carbonate, sodium dodecyl sulfate (SDS), β-mercaptoethanol (2-ME), and Coomassie brilliant blue R-250 were purchased from Carl Roth (Karlsruhe, Germany). Precast gels (Any kD Mini-PROTEAN TGX Gels, 15 well, 15 μL) and sample loading buffer (4× Laemmli Sample Buffer) were purchased from Bio-Rad (Mü nchen, Germany). Molecular weight marker (Mark12 Unstained Standard) was purchased from Life Technologies (Karlsruhe, Germany). Water (H2O) was prepared on a Milli-Q gradient A10 system by Millipore (Schwalbach, Germany) and used for all buffers. Received: April 1, 2015 Revised: May 24, 2015 Accepted: June 3, 2015
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DOI: 10.1021/acs.jafc.5b01634 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effect of buffer system on 2S extraction. (A) Purity of 2S albumin extracts from mustard given as percentage of the total extracted protein in different buffer systems. Superscript letters indicate significance groups (p ≤ 0.05): 1, glycine (100 mM, pH 2.5); 2, acetate (100 mM, pH 3.6); 3, citrate (100 mM, pH 4.0); 4, citrate (100 mM, pH 5.0); 5, MES (100 mM, pH 6.0); 6, MOPS (100 mM, pH 7.0); 7, Tris (100 mM, pH 8.0); 8, glycine (100 mM, pH 9.0); 9, bicarbonate (100 mM, pH 11.0); 10, Millipore H2O. (B) Representative SDS-PAGE gel. Numbering is according to (A). Preparation of Defatted Seed and Nut Flour. All seeds and nuts containing more than ∼40% fat (sunflower seeds, hazel nuts, pistachio seeds, Brazil nut seeds, sesame seeds, pecan seeds, cashew seeds, walnuts, and peanut seeds) were defatted twice prior to analysis. Seeds and nuts were crushed for 2−3 s in a knife mill (Grindomix GM 100, Retsch, Haan, Germany), resulting in a coarse flour. The first defatting step was performed by adding n-pentane (1 g/10 mL) and continuous stirring for 2 h at room temperature. The slurry was sieved and air-dried overnight. All seeds and nuts containing 90% and no significant differences between these three buffer systems. All remaining buffers gave only poor 2S albumin purities between 9.8 and 51.0% and a strongly reduced yield as demonstrated by SDS-PAGE (Figure 1B). Notably, the lowest purity and yield were observed for water (Figure 1, sample 10). This is of particular interest, as current methods in place for 2S albumin isolation mainly use water for the extraction. Garino et al. purified hazelnut 2S albumins by D
DOI: 10.1021/acs.jafc.5b01634 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry routines, an intermediate buffer molarity of 100 mM was chosen for further experiments. Effect of pH. As the pH of the buffer system was the most critical parameter in the initial experiment, we further assessed the influence of different pH values for the three most promising acidic buffers (glycine, acetate, and citrate) at molar buffer concentrations of 100 mM and 60 °C. Extraction of 2S albumins was analyzed for the three buffers over the respective buffering pH range in steps of ∼0.5 pH unit. Results are summarized in Table S1 and Figure 4. For the glycine buffer, an increase in pH to 3.5 resulted in reduced purity compared to extraction at pH 2.5 or 3.0. The citrate buffer showed equal purity of extracted 2S albumins over the complete pH range between 2.5 and 4.0. In contrast, an increase of pH of the acetate buffer from 3.6 to 4.0 led to a significant decrease in purity. Optimized Buffer Conditions. We demonstrate that not a single optimal buffer system exists, but the extraction must comply with certain conditions: In our approach the most important parameter is the acidic pH of the buffer system and consequently glycine, acetate, or citrate buffers that have optimal buffering capacity between pH 2.5 and 4.0 resulted in the highest 2S albumin purity and high protein yield. The extraction temperature had no significant influence on purity, but elevated temperatures resulted in increased 2S albumin yield. This is especially interesting as 2S albumins are highly thermostable.9 Optimal results with respect to buffer molarity are obtained in a broad range between 20 and 500 mM. Notably, the purity of 2S extracts is reduced when buffer molarity is further increased. Performance Control and Transferability. As the optimized acidic buffer conditions are not common for protein extraction, we analyzed the secondary structure of the acidic protein extract and compared it to a 2S albumin fraction extracted in 100 mM Tris-HCl, pH 8.0, and purified by SEC. Extractions with three different acidic buffer systems (100 mM glycine, pH 2.5, 60 °C; 100 mM acetate, pH 3.6, 60 °C; and 100 mM citrate, pH 4.0, 60 °C) were compared with the SECpurified 2S albumins using CD spectroscopy. To eliminate the influence of different buffers on CD spectra, a buffer exchange step to 12 mM phosphate buffer was included for all samples prior to CD spectroscopy. For all samples, we obtained CD spectra typical for 2S albumins (which mainly consist of αhelical structures), and no significant differences between the acidic extracts and the SEC-purified 2S albumins extracted with Tris buffer were observed (Figure 5), demonstrating that acidic extraction conditions do not interfere with 2S albumin structure. As it is intended to provide a method generally applicable for the extraction of 2S albumins, we assessed the transferability from mustard to further seeds and nuts (see Materials and Methods). For 9 of 13 matrices included in this study, we observed excellent purity of 2S albumin extract and very good yield as exemplified in Figure 6. The crude protein was extracted with 100 mM Tris-HCl, pH 8.0, 23 °C (with an addition of 2 M NaCl for extraction of walnut according to the method of Sathe et al.13) and was compared to the extraction with 100 mM glycine, pH 2.5, 60 °C. Notably, selective 2S albumin extraction was not obtained for food matrices from legumes (peanut, chickpea, and soybean), and sunflower showed significantly reduced selectivity (Figure 6). As we did not observe phylogenetic clustering of extractable and “nonextractable” 2S albumin sequences (data not shown), we
Figure 5. Circular dichroism spectroscopy of acidic buffers (glycine, acetate, and citrate) at 60 °C and 2S albumin preparation purified with GPC after Tris extraction at 20 °C. No significant differences in spectra are observed, and all obtained spectra are typical for 2S albumins, which are rich in α-helical secondary structure.
assume that differences in food matrices underlie reduced selectivity of the extraction for peanut, chickpea, soybean, and sunflower 2S albumins. In our study we present a simple, rapid, and selective method for the isolation of 2S albumins from different seeds and nuts. We achieved 2S albumin purities up to 99% and excellent yield for our model protein Sin a 1 from mustard seeds and demonstrate excellent transferability to other allergenic seeds and nuts. In comparison to conventional chromatographybased purification methods (IEX, SEC, RP-HPLC), the newly developed method requires only minimal laboratory equipment and allows in principle unlimited protein yield with higher protein concentration compared to currently used chromatographic methods. The purification of 2S albumins is relevant for different fields of interest in allergen research. With the selective extraction presented in this study, the production of allergen extracts from different natural sources that contain a single allergen with its natural variants and isoforms is feasible. Due to the pronounced stability of 2S albumins, neither acidic pH nor elevated extraction temperature influences the structure as demonstrated by CD spectroscopy. Consequently, component-resolved diagnostic (CRD) of natural allergens might become possible. Today, allergen preparations for CRD are derived from recombinant expression as crude allergen extracts from natural sources are too complex and might not be reproducible.14 Using selective extraction of 2S albumins results in the high yield of an isolated allergen with its isoforms and variants that might be employed (after a further purification step if necessary) for CRD. Although crude natural extracts are too complex for allergen diagnosis, isolated recombinant single isoforms of allergens do not reflect the natural complexity of allergens such as 2S albumins. Selective extraction might be an alternative for 2S albumins. It has to be noted that we did not investigate the immunological properties of the 2S extracts obtained in this study, which would be an essential prerequisite when aiming to use such extracts for CRD. Trace amounts of residual further allergens such as 11S legumins or 7S vicilins might influence the immunological properties, and this interference has to be ruled out. However, the high yield of 2S allows for efficient further downstream purification if necessary. E
DOI: 10.1021/acs.jafc.5b01634 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 6. Transfer of optimized 2S extraction conditions to further seed and nut matrices. Total protein extract is performed with T = Tris-HCl (100 mM, pH 8.0, 23 °C), and 2S-selective extraction is done with G = glycine (100 mM, pH 2.5, 60 °C), respectively. (A) Allergenic foods from which 2S albumins are efficiently and selectively extracted: 1, buckwheat-T; 2, buckwheat-G; 3, cashew-T; 4, cashew-G; 5, hazelnut-T; 6, hazelnut-G; 7, Brazil nut-T; 8, Brazil nut-G; 9, pecan nut-T; 10, pecan nut-G; 11, pistachio-T; 12, pistachio-G; 13, sesame-T; 14, sesame-G; 15, walnut-T; 16, walnut-G. (B) Foods from which 2S albumins are not sufficiently extracted. Note that the SDS-PAGE conditions are not optimized to separate 2S albumin subunits.
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English walnut (Juglans regia). J. Agric. Food Chem. 2014, 62, 11767− 11775. (7) Menendez-Arias, L.; Moneo, I.; Dominguez, J.; Rodriguez, R. Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a 1. Eur. J. Biochem. 1988, 177, 159−166. (8) Liu, C.; Wang, H.; Cui, Z.; He, X.; Wang, X.; Zeng, X.; Ma, H. Optimization of extraction and isolation for 11S and 7S globulins of soybean seed storage protein. Food Chem. 2007, 102, 1310−1316. (9) Moreno, F. J.; Maldonado, B. M.; Wellner, N.; Mills, C. Thermostability and in vitro digestibility of a purified major allergen 2S albumin (Ses i 1) from white sesame seeds (Sesamum indicum L.). Biochim. Biophys. Acta, Proteins Proteomics 2005, 1752, 142−153. (10) Garino, C.; Zuidmeer, L.; Marsh, J.; Lovegrove, A.; Morati, M.; Versteeg, S.; Schilte, P.; Shewry, P.; Arlorio, M.; van Ree, R. Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut. Mol. Nutr. Food Res. 2010, 54, 1257−1265. (11) Galbas, M.; Porzucek, F.; Woźniak, A.; Słomski, R.; Selwet, M. Isolation of low-molecular albumins of 2S fraction from soybean (Glycine max (L.). Acta Biochim. Polym. 2013, 60, 107−110. (12) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (13) Sathe, S. K.; Venkatachalam, M.; Sharma, G. M.; Kshirsagar, H. H.; Teuber, S. S.; Roux, K. H. Solubilization and electrophoretic characterization of select edible nut seed proteins. J. Agric. Food Chem. 2009, 57, 7846−7856. (14) Valenta, R.; Lidholm, J.; Niederberger, V.; Hayek, B.; Kraft, D.; Grönlund, H. The recombinant allergen-based concept of componentresolved diagnostics and immunotherapy (CRD and CRIT). Clin. Exp. Allergy 1999, 29, 896−904.
ASSOCIATED CONTENT
S Supporting Information *
Figure S1 and Table S1. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01634.
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AUTHOR INFORMATION
Corresponding Author
*(J.B.) Phone: +49 251 8333392. Fax: +49 251 833396. E-mail:
[email protected]. Funding
M.H. was supported by a grant from the Heinrich-StockmeyerStiftung, Bad Rothenfelde. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Katrin Bassen for the excellent preparation of food photographs used in the table of contents (TOC).
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REFERENCES
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