A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen.
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.
Best
E A
EA
Heska Corporation, Fort Collins, Colorado 80525, USA. beste@heska.com
Stedman
K E
KE
Bozic
C M
CM
Hunter
S W
SW
Vailes
L
L
Chapman
M D
MD
McCall
C A
CA
McDermott
M J
MJ
D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides; D002241:Carbohydrates / Q000032:analysis; D003001:Cloning, Molecular / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D013058:Mass Spectrometry; D008925:Mites; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D000485:Allergens; D039741:Antigens, Dermatophagoides; D002241:Carbohydrates; D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D006026:Glycoside Hydrolases; D010450:Endopeptidases