High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.
BACKGROUND
The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success.


OBJECTIVE
The aim of this study was to establish an efficient system for the production of recombinant Der p 1.


METHODS
The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity.


RESULTS
ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1.


CONCLUSIONS
This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.
Jacquet
A
A
Department of Applied Genetics, Université Libre de Bruxelles, Belgium. ajacquet@sga.ulb.ac.be
Magi
M
M
Petry
H
H
Bollen
A
A
D039741:Antigens, Dermatophagoides / Q000096:biosynthesis* / Q000276:immunology; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; D006636:Histamine Release; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1