Biochemical and Biophysical Research Communications
Crystal structure and some properties of a major house dust mite allergen, Derf 2
Section snippets
Materials and methods
Preparation of Derf 2 and its conformational variant. Recombinant Derf 2 protein was prepared as described previously [19]. In brief, Escherichia coli Rosseta(DE3)pLys (Merck KgaA, Darmstadt, Germany) was transfected with an expression plasmid harboring cDNA for Derf 2 and induced for protein expression with 1 mM IPTG. The resultant inclusion bodies were dissolved in buffer containing 8 M urea and 1 M guanidine, and refolded in arginine-based buffer. After dialysis against 10 mM Tris–HCl buffer, pH
Crystallization and structure determination
In search of hypoallergenic variants of Derf 2 for the use in hyposensitization therapy for allergic asthma, we previously demonstrated that the structure of allergen is intrinsically involved in allergenicity. Since only preliminary X-ray data were available [30], we first attempted to prepare crystals of Derf 2 and solve the X-ray structure in the present study. As shown in Fig. 1A, Derf 2 was crystallized in a mixture consisting of 20 mg/ml Derf 2, 23% PEG4000, 0.06 M ammonium sulfate, 0.1 M
Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology, Japanese Government, the Inamori Foundation, Sumitomo Pharmaceutical Co., Ltd., and the Foundation for Biomedical Research and Innovation. An expression plasmid encoding cDNA for Derf 2 was kindly provided by Dr. Toyokazu Yokota, Asahi Breweries, Tokyo, Japan.
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