The Disulphide Mapping, Folding and Characterisation of Recombinant Ber e 1, an Allergenic Protein, and SFA8, Two Sulphur-rich 2S Plant Albumins

Abstract

We have cloned and expressed genes encoding the allergenic brazil nut 2 S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2 S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphurrich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.

Differential Polarization of Immune Responses by Plant 2S Seed Albumins, Ber e 1, and SFA8

“…The tertiary structure of the SFA8 in solution has been determined recently (24) and agrees with the tertiary structures of other members of the family and the model structure proposed for Ber e 1 (21). This conservation of the three-dimensional structure was exploited in the design of chimeric proteins for the mapping of the human structural IgE epitope of Ber e 1, whereby proteins containing exchanged domains were produced (25).…”

“…The reverse-phase purified recombinant proteins were shown to be absent of endotoxin, as determined by an Endosafe Kit (Charles River Laboratories). Our previous results have unequivocally established that the recombinant proteins mimic their native counterparts in structural and biophysical properties (21)(22)(23). Recent nuclear magnetic resonance results (21) and disulfide mapping by mass spectra (21) have shown that the four intrachain disulfide bridges were properly established during the protein expression, folding, and secretion in the yeast expression system used.…”

“…Recombinant Brazil nut and sunflower seed 2S albumins (Ber e 1 and SFA8) were produced as secreted full-length proteins by the yeast Pichia pastoris and purified by FPLC using a heparin-Sepharose column, as previously described (21). Both single SDS-PAGE band proteins (under denaturing conditions) were further purified as single peaks by preparative reverse-phase chromatography also as previously described (25).…”

“…The native and the recombinant forms of both Ber e 1 and SFA8 have previously been shown to possess similar stability to pepsin-containing simulated gastric fluid (21,22) and also resistance to high temperatures, guanidinium chloride, and acidic pH conditions (22,23). Therefore, the stability of these proteins does not appear to account for their different allergenicities.…”

Seed storage albumins: biosynthesis, trafficking and structures

“…CysV and CysVI are also highly conserved and flank a single residue whereas CysVII and CysVIII vary in their position and may be separated by 3-7 residues. Although (Rundqvist et al 2012) (a) (b) (Irwin et al 1990;Alcocer et al 2002;Pantoja-Uceda et al 2004a). The structures include a recombinant Brassica napus albumin (rproBnIb) that differs from the native BnIb protein by three residues (black) that join the small and large albumin subunit.…”