Expression of the house dust mite allergen Der p 2 in the baker's yeast Saccharomyces cerevisiae.
RESULTS
The major house dust mite allergen Der p 2 was expressed as a recombinant mature protein in the baker's yeast Saccharomyces cerevisiae. The yeast produces the protein fused to the invertase signal peptide, leading to the secretion of Der p 2 as a soluble protein into the culture medium. The signal peptide is hereby cleaved off, resulting in a mature allergen. In this system Der p 2 was produced in 7.6 (+/-2.9) mg/L growth culture. Purification of the recombinant allergen was achieved by a single gel filtration step, resulting in a purity > or = 95%. The yeast-derived Der p 2 was almost indistinguishable from natural Der p 2 with respect to IgE-reactivity and binding to the majority of Der p 2 specific MoAbs -- as was shown in RAST analysis (n = 168) and a sandwich ELISA and RIA analysis, respectively. Recombinant and natural Der p 2 also showed similar biological activity in histamine release assays (n = 4).


CONCLUSIONS
An expression system for Der p 2 was developed that enables the production of a soluble allergen in the culture supernatant with immunological characteristics similar to the natural allergen. In addition, yeast offers the advantage of the absence of endotoxin in comparison to E. coli. This might facilitate acceptance of recombinant allergens for in vivo applications as immunotherapy or skin-prick testing.
Hakkaart
G A
GA
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and the Laboratory for Experimental and Clinical Immunology of the University of Amsterdam.
Harmsen
M M
MM
Chua
K Y
KY
Thomas
W R
WR
Aalberse
R C
RC
Van Ree
R
R
D000485:Allergens / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology; D039741:Antigens, Dermatophagoides; D017077:Culture Media, Conditioned / Q000737:chemistry; D015870:Gene Expression / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006636:Histamine Release / Q000187:drug effects; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D008925:Mites / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000276:immunology; D017421:Sequence Analysis
D000485:Allergens; D000911:Antibodies, Monoclonal; D039741:Antigens, Dermatophagoides; D017077:Culture Media, Conditioned; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E