[en] 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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Alcocer, Marcos
Murtagh, G. J.
Bailey, Kevin
Dumoulin, Mireille ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
Meseguer, A. S.
Parker, Martin
Archer, David
Language :
English
Title :
The Disulphide Mapping, Folding and Characterisation of Recombinant Ber e 1, an Allergenic Protein, and SFA8, Two Sulphur-rich 2 S Plant Albumins
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