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

摘要

We have cloned and expressed genes encoding the allergenic brazil nut2 S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophicyeast Pichia pastoris. We show that both proteins were secretedat high levels and that the purified proteins were properly folded. Wealso showed that Ber e 1 is glycosylated during secretion and that theglycan does not interfere with the folding or immunoreactivity. Thedisulphide map of the Ber e 1 protein was experimentally established andis in agreement with the conserved disulphide structure of other membersof the 2 S albumin family. A model three-dimensional structure of theallergen was generated. During the expression studies and throughmutation we have also shown that alteration of the sequences around theKex2 endoproteolytic processing site in the expressed fusion protein cancompromise the secretion by targeting part of the protein for possibledegradation. The secreted production of these properly folded sulphurrichplant albumins presents an opportunity to delineate the attributesthat make an allergen and to facilitate the diagnosis and therapy of typeI allergy.