A Recombinant Group 1 House Dust Mite Allergen, rDer f 1, with Biological Activities Similar to Those of the Native Allergen

• Published in: Protein expression and purification 2000-12, Vol.20 (3), p.462-471
• Main Authors: Best, Elaine A., Stedman, Kim E., Bozic, Cynthia M., Hunter, Shirley Wu, Vailes, Lisa, Chapman, Martin D., McCall, Catherine A., McDermott, Martin J.
• Format: Article
• Language: English
• Subjects: Allergens - chemistry; Allergens - genetics; Allergens - immunology; Amino Acid Sequence; Animals; Antigens, Dermatophagoides; Carbohydrates - analysis; Cloning, Molecular - methods; Endopeptidases - metabolism; Enzyme-Linked Immunosorbent Assay; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - immunology; Glycoside Hydrolases - metabolism; Humans; Immunoglobulin E - immunology; Mass Spectrometry; Mites; Molecular Sequence Data; Pichia - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.2000.1327

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an ∼25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae α factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35–50 kDa. Both the α factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, ∼25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.