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Expanded bed adsorption as a fast technique for the large-scale purification of the complete isoform pool of Ber e 1, the major allergen from Brazil nuts

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Author: Boxtel, E.L. van · Koningsveld, G.A. van · Koppelman, S.J. · Broek, L.A.M. van den · Voragen, A.G.J. · Gruppen, H.
Institution: TNO Kwaliteit van Leven
Source:Molecular Nutrition and Food Research, 3, 50, 275-281
Identifier: 239143
doi: doi:10.1002/mnfr.200500203
Keywords: Biology · Biotechnology · Allergen · Ber e 1 · Chromatography · Expanded bed adsorption · Purification · 2S albumin precursor, Bertholletia excelsa · albuminoid · allergen · protein precursor · vegetable protein · adsorption · article · Brazil nut · chemistry · chromatography · circular dichroism · isolation and purification · mass spectrometry · methodology · plant seed · polyacrylamide gel electrophoresis · Adsorption · Albumins · Allergens · Bertholletia · Chromatography · Circular Dichroism · Electrophoresis, Polyacrylamide Gel · Plant Proteins · Protein Precursors · Seeds · Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization · Bertholletia excelsa · Human echovirus 1 · Ziziphus mauritiana


A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Bere 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1. © 2006 WILEY-VCH Verlag GmbH & Co. KGaA.