Best E A, Stedman K E, Bozic C M, Hunter S W, Vailes L, Chapman M D, McCall C A, McDermott M J
Heska Corporation, Fort Collins, Colorado 80525, USA.
Protein Expr Purif. 2000 Dec;20(3):462-71. 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 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.
针对粉尘螨粪便中发现的一种蛋白酶Der f 1的血清IgE，与人类对屋尘螨的过敏致敏密切相关，并且是发生哮喘的一个危险因素。天然Der f 1（nDer f 1）以前体-前体形式产生，并加工成约25 kDa的成熟形式。我们使用AOX1启动子表达载体在毕赤酵母中表达了Der f 1的重组形式（rDer f 1）。将酶原形式或成熟形式与酿酒酵母α因子前体-前体序列融合，导致该蛋白的成熟形式从毕赤酵母中分泌出来。分泌的蛋白在单个N-糖基化位点进行了异质性糖基化，表观分子量为35-50 kDa。α因子信号肽和酶原区域在分泌过程中均被有效加工。一种具有突变的共有N-连接糖基化位点的酶原形式从毕赤酵母中分泌出来，成为一种成熟的、未糖基化的、约25 kDa的蛋白。这种未糖基化的rDer f 1的IgE结合活性与毕赤酵母产生的糖基化形式以及从螨虫中获得的nDer f 1相似。因此，寡糖对于从毕赤酵母中分泌或体外IgE结合不是必需的。Der f 1的重组形式和天然形式在酪蛋白酶谱凝胶上均显示出蛋白酶活性。高纯度重组Der f 1的可得性将有助于螨虫过敏的实验和临床研究。
