Format

Send to

Choose Destination
See comment in PubMed Commons below
Int Arch Allergy Immunol. 2005 May;137(1):1-8. Epub 2005 Mar 21.

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.

Author information

1
Atopy (Allergy) Research Center, Juntendo University School of Medicine, Hongo, Tokyo Japan. t-takai@med.juntendo.ac.jp

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.

PMID:
15785076
DOI:
10.1159/000084607
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for S. Karger AG, Basel, Switzerland
    Loading ...
    Support Center