A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen.

Abstract

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 approximately 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 alpha 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 alpha 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, approximately 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.

• Authors: E A Best, K E Stedman, C M Bozic, S W Hunter, L Vailes, M D Chapman, C A McCall, M J McDermott
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 20 Issue 3 Pg. 462-71 (Dec 2000) ISSN: 1046-5928 [Print] United States
• PMID: 11087686 (Publication Type: Journal Article)
• Copyright: Copyright 2000 Academic Press.

Chemical References

• Allergens
• Antigens, Dermatophagoides
• Carbohydrates
• Glycoproteins
• Recombinant Proteins
• Immunoglobulin E
• Glycoside Hydrolases
• Endopeptidases

Topics

• Allergens (chemistry, genetics, immunology)
• Amino Acid Sequence
• Animals
• Antigens, Dermatophagoides
• Carbohydrates (analysis)
• Cloning, Molecular (methods)
• Endopeptidases (metabolism)
• Enzyme-Linked Immunosorbent Assay
• Glycoproteins (chemistry, genetics, immunology)
• Glycoside Hydrolases (metabolism)
• Humans
• Immunoglobulin E (immunology)
• Mass Spectrometry
• Mites
• Molecular Sequence Data
• Pichia (genetics)
• Recombinant Proteins (chemistry, genetics, immunology)