Cloning and Expression of cDNA Encoding the Complete Prepro-Form of an Isoform of Der f 1, the Major Group 1 Allergen from House Dust Mite Dermatophagoides farinae(1
The major house dust mite allergens are classified into group 1 (Der f 1 and Der p 1) or group 2 (Der f 2 and Der p 2). Der f 1 and Der pi, which belong to the papain-like cysteine protease family, are mainly found in mite feces, and are the most important allergens clinically and in standardization of mite extract. The cDNA encoded the amino acid sequence containing a putative signal sequence from – 98 to – 81, a putative prosequence from – 80 to – 1, and a mature sequence from 1 to 223. A sequence comparison among the prepro-forms of three house dust mite group 1 allergens, Der f 1 from D. farinae was made with the sequences reported by Der pI from D. pteronyssinus, and Eur m 1 from Euroglyphus maynei.
Pro-forms of rDer f 1, the wild-type and N53Q, were secreted into the medium of P. pastoris. The wild-type Der f 1 has an N-glycosylation motif conserved among mite group 1 allergens. In N53Q, the N-glycosylation motif is disrupted by sitedirected mutagenesis. The prosequence was removed by the in vitro activation.
The mature wild-type Der f 1 and N53Q, which were expressed as the pro-forms in P. pastoris and then converted to the mature forms, had IgE binding equivalent to natural Der f 1. On the other hand, the IgE-binding capacity of the polypeptide of the mature wild-type Der f 1 directly expressed in E. coli had low IgE-binding.
N-glycosylation did not affect IgE-binding of rDer f 1, which was expressed as the pro-form in P. pastoris and then converted to the mature form. Therefore, the failure of rDer f 1 directly expressed in E. coli to bind IgE might result from the absence of the pro sequence in the refolding process or because the conditions for refolding were not optimized. Maturation of IgE-binding and protease activity of rDer f 1, which was expressed as the pro-form in E. coli and then converted to the mature form. The pro sequence might be essential for refolding of Der f 1 in these systems.