title: Large-scale production of major house dust mite allergen der f 2 mutant (C8/119S) in Escherichia coli.
abstract: Hyposensitization, in which causative antigens of allergic diseases are injected, is the sole means of a radical cure for allergic diseases. Since the therapeutic allergens currently used are naturally extracted, producing preparations with a stable titer from such extracts is extremely difficult. There are several reports on the expression of recombinant mite allergens in Escherichia coli using inducers. The use of an inducer for industrial production will lead to high costs and, for therapeutic use, it must be removed in the purification process. C8/119S is a mutant of Der f 2, a major house dust mite allergen. The C8/119S gene was integrated downstream of the trp promoter to produce the expression plasmid (pWU11-C8/119S). Then, this expression plasmid was used to transform E. coli strain HB101 (pWU11-C8/119S/HB101). A recombinant E. coli pWU11-C8/119S/HB101 did not express C8/119S in a low-temperature culture (32 degrees C), but C8/119S was induced to a high level of expression in a high-temperature culture (37 degrees C). pWU11-C8/119S/HB101 proliferated when expression was induced by high temperature and an approximately 3-fold greater proliferation was obtained compared with the use of an inducer in a large-scale culture. The C8/119S protein was expressed as inclusion bodies and obtained by refolding and chromatography purifications. The immunological properties of C8/119S were assessed by western blotting. Western blotting demonstrated that purified C8/119S reacted with a monoclonal anti-Der f 2 antibody (18G8). pWU11-C8/119S/HB101 can be used as an easy, low cost expression system on a large scale. It is also advantageous for industrial production in that the addition of an inducer is not required to achieve expression of the mite allergen.
authors.affiliation: Applied Research Department, The Chemo-Sero-Therapeutic Research Institute (Kaketsuken), 1314-1 Kyokushikawabe, Kikuchi, Kumamoto 869-1298, Japan. koyanagi@kaketsuken.or.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Koyanagi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Maeda
authors.forename: Toshio
authors.initials: T
authors.lastname: Murakami
authors.forename: Kenjirou
authors.initials: K
authors.lastname: Kawatsu
authors.forename: Keishin
authors.initials: K
authors.lastname: Sugawara
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Miyatsu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mizokami
mesh_terms: D000485:Allergens; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry / Q000378:metabolism*; D060829:Arthropod Proteins; D001709:Biotechnology / Q000379:methods*; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D006967:Hypersensitivity / Q000378:metabolism* / Q000628:therapy; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451160:Dermatophagoides farinae antigen f 2