The disulphide mapping, folding and characterisation of recombinant Ber e 1, an allergenic protein, and SFA8, two sulphur-rich 2S plant albumins.
We have cloned and expressed genes encoding the allergenic brazil nut 2S 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 2S 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 sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.
Alcocer
Marcos J C
MJ
School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. marcos.alcocer@nottingham.ac.uk
Murtagh
Gareth J
GJ
Bailey
Kevin
K
Dumoulin
Mireille
M
Meseguer
Amparo Sarabia
AS
Parker
Martin J
MJ
Archer
David B
DB
D055516:2S Albumins, Plant; D000418:Albumins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000485:Allergens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003429:Cross Reactions; D004220:Disulfides / Q000737:chemistry; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009754:Nuts / Q000737:chemistry / Q000235:genetics; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D043484:Proprotein Convertases; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000378:metabolism; D013455:Sulfur
D055516:2S Albumins, Plant; D000418:Albumins; D000485:Allergens; D052179:Antigens, Plant; D004220:Disulfides; D010940:Plant Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C067568:methionine-rich 2S protein, Helianthus; C103364:2S albumin, brazil nut; D013455:Sulfur; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae