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

被引:39
作者
Best, EA
Stedman, KE
Bozic, CM
Hunter, SW
Vailes, L
Chapman, MD
McCall, CA
McDermott, MJ
机构
[1] Heska Corp, Ft Collins, CO 80525 USA
[2] Univ Virginia, Dept Med, Asthma & Allerg Dis Ctr, Charlottesville, VA 22908 USA
关键词
D O I
10.1006/prep.2000.1327
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 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 cu 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 cu 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, similar to 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. (C) 2000 Academic Press.
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页码:462 / 471
页数:10
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