title: Dilution method to refold bacterially expressed recombinant Der f 2 and Der p 2 to exhibit the secondary structure and histamine-releasing activity of natural allergens.
abstract: BACKGROUND
  Structurally refolded recombinant forms of major house dust mite group 2 allergens, Der f 2 and Der p 2, expressed in Escherichia coli, were prepared by solubilizing the insoluble products with urea and subsequently dialyzing against buffer. In this study, we determined conditions for refolding the urea-denatured recombinant Der f 2 and Der p 2 by one-step dilution as an alternative to dialysis, which requires several steps of handling and much time and cost.
  
  
  METHODS
  The insoluble bacterial product containing recombinant Der f 2 was solubilized with a buffer containing 8 M urea, and the solution was diluted to various urea concentrations. The refolding efficiency in each dilution was estimated from the height of the peak corresponding to the folded recombinant Der f 2 and that containing the aggregated form on anion exchange chromatography. The structure and allergenicity of the purified recombinant Der f 2 and Der p 2 refolded using the dilution method were analyzed based on circular dichroism and a basophil histamine-releasing assay, respectively.
  
  
  RESULTS
  Although the refolding efficiency decreased as the urea concentration in the dilution increased, experimental conditions whereby the protein and urea concentrations in the dilution were less than 0.5 mg/ml and 0.8 M, respectively, achieved maximum refolding efficiency. The recombinant allergens prepared by the dilution method exhibited the secondary structure and histamine-releasing activity of natural allergens purified from mite culture.
  
  
  CONCLUSIONS
  The dilution method established in this study is more convenient in terms of handling, time, and cost than the dialysis method and will be useful for large-scale production and for the preparation of numbers of mutants to analyze IgE epitopes.
authors.affiliation: Atopy (Allergy) Research Center, Juntendo University School of Medicine, Hongo, Tokyo Japan. t-takai@med.juntendo.ac.jp
authors.forename: Toshiro
authors.initials: T
authors.lastname: Takai
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Takaoka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yasueda
authors.forename: Ko
authors.initials: K
authors.lastname: Okumura
authors.forename: Hideoki
authors.initials: H
authors.lastname: Ogawa
mesh_terms: D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D060829:Arthropod Proteins; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006636:Histamine Release / Q000276:immunology; D017510:Protein Folding; D017433:Protein Structure, Secondary; D039981:Pyroglyphidae / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451160:Dermatophagoides farinae antigen f 2; C451163:Dermatophagoides pteronyssinus antigen p 2; D011994:Recombinant Proteins; D004247:DNA