Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding.
BACKGROUND
Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed.


OBJECTIVE
Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1.


METHODS
The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively.


RESULTS
The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities.


CONCLUSIONS
The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.
Yasuhara
T
T
Bioscience Research and Development Laboratory, Asahi Breweries Ltd, 1-21, Midori 1-chome, Moriya-machi, Kitasoma-gun, Ibaraki, Japan.
Takai
T
T
Yuuki
T
T
Okudaira
H
H
Okumura
Y
Y
D000818:Animals; D039741:Antigens, Dermatophagoides; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004391:Dust / Q000009:adverse effects; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006798:Housing; D006969:Hypersensitivity, Immediate / Q000175:diagnosis*; D007073:Immunoglobulin E / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008925:Mites / Q000276:immunology*; D008969:Molecular Sequence Data; D011852:Radioallergosorbent Test; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D039741:Antigens, Dermatophagoides; D004391:Dust; D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases