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.
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, Ber e 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1.
van Boxtel
Evelien L
EL
Centre for Protein Technology, Wageningen, The Netherlands.
van Koningsveld
Gerrit A
GA
Koppelman
Stef J
SJ
van den Broek
Lambertus A M
LA
Voragen
Alfons G J
AG
Gruppen
Harry
H
D055516:2S Albumins, Plant; D000327:Adsorption; D000418:Albumins / Q000302:isolation & purification*; D000485:Allergens / Q000302:isolation & purification*; D052179:Antigens, Plant; D031383:Bertholletia / Q000737:chemistry; D002845:Chromatography / Q000379:methods*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D010940:Plant Proteins / Q000302:isolation & purification*; D011498:Protein Precursors / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D055516:2S Albumins, Plant; D000418:Albumins; D000485:Allergens; D052179:Antigens, Plant; D010940:Plant Proteins; D011498:Protein Precursors; C103364:2S albumin, brazil nut