title: Production of enzymatically and immunologically active Der f 1 in Escherichia coli.
abstract: Der f 1 is a major house dust mite allergen belonging to the cysteine protease family. Because of the great demand for clinical and research use of this allergen, much effort to establish an efficient method of preparing purified Der f 1 has been made. We constructed an isopropyl-beta-D-thiogalactopyranoside-inducible expression plasmid to produce the pro-form of Der f 1 in Escherichia coli. The recombinant product was accumulated as insoluble inclusion bodies in cells. The solubilized inclusion bodies were found to be successfully renatured by two-step gel filtration chromatography. About 70 mg of pro Der f 1 were properly refolded by this method from 1 liter of culture. Acid treatment of the renatured pro Der f 1 resulted in the autocatalytic removal of the pro-sequence. The obtained mature form of Der f 1 bound IgE in patient sera and induced the release of histamine from peripheral blood leukocytes equally to native Der f 1. Furthermore, mature Der f 1 obtained by this method had identical protease activity with native Der f 1. We also discussed the contribution of the pro-sequence and the sugar chain of Der f 1 to its antigenic and enzymatic activity. This is the first report to produce an active mature form of recombinant Der f 1 in E. coli.
authors.affiliation: Foods and Pharmaceuticals Research and Development Laboratory, Asahi Breweries Ltd., Ibaraki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
authors.forename: T
authors.initials: T
authors.lastname: Takai
authors.forename: T
authors.initials: T
authors.lastname: Yasuhara
authors.forename: T
authors.initials: T
authors.lastname: Yuuki
authors.forename: Y
authors.initials: Y
authors.lastname: Ohtake
authors.forename: T
authors.initials: T
authors.lastname: Yokota
authors.forename: Y
authors.initials: Y
authors.lastname: Okumura
mesh_terms: D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006636:Histamine Release / Q000187:drug effects; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism; D008925:Mites / Q000276:immunology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D003546:Cysteine Endopeptidases