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.

Application of Immunoreaction Enhancer Solutions to an Enzyme-Linked Immunosorbent Assay for Antigen-Specific IgE in Mice Immunized with Recombinant Major Mite Allergens or Ovalbumin

“…An ELISA simply using the enhancer solutions, which obtains remarkably strengthened signals for allergen-specific IgE, would be useful for the study of allergies with murine models. In addition, this paper showed that recombinant forms of the major mite allergens Der p 1, Der f 1 [12,13,14,15,16,17, 23] and Der f 2 [18,19,20, 24] can elicit a significant allergen-specific IgE response in mice.…”

“…As the B cell epitopes for recombinant forms with and without hyperglycosylation are considered to be equivalent [12, 13], we used the hyperglycosylated Der p 1-WT and Der f 1-WT for detecting Der p 1- and Der f 1-specific Abs as alternatives to the unglycosylated rDer p 1 and rDer f 1, whose production is much lower than that of the hyperglycosylated forms in yeast [16]. Recombinant Der f 2 (rDer f 2) was prepared as previously described [18] and used for immunization and the coating of microtiter plates for ELISA for Der f 2-specific Abs.…”

“…In the present study, we evaluated the effects of in-well pretreatment of sera with protein G-coupled sepharose beads and the use of enhancer solutions on signals for the binding of allergen-specific IgE to plates coated with recombinant forms of the clinically relevant major house dust mite allergens [8,9,10,11] Der p 1, Der f 1 [12,13,14,15,16,17] and Der f 2 [18,19,20], or with OVA. We demonstrate that both methods are effective and that especially the latter has remarkable advantages in terms of signal intensity and handling.…”

Modulation of Allergenicity of Major House Dust Mite Allergens Der f 1 and Der p 1 by Interaction with an Endogenous Ligand

“…48 and 49), and a recombinant form of a mite major group 2 allergen, Der f 2 (50). A precursor form of rDer f 1-C35S was prepared by site-directed mutagenesis, secreted into the yeast culture supernatant, and converted to its mature form by yeast-derived protease activity within the supernatant by dialysis with an acidic buffer (48).…”

“…After the dilution, the concentration of cystatin A was 500 ng/ml. The method used to measure the amount of histamine released from basophils was as described previously (19,50). Effective concentrations yielding half-maximal allergen response were determined from curves in the histamine-releasing assay by using Prism version 4.0 (GraphPad).…”