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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/29275
Title: Structural and IgE binding analyses of recombinant Der p 2 expressed from the hosts Escherichia coli and Pichia pastoris
Authors: S. Tanyaratsrisakul
N. Malainual
O. Jirapongsananuruk
W. A. Smith
W. R. Thomas
S. Piboonpocanun
Mahidol University
Telethon Kids Institute
Keywords: Immunology and Microbiology;Medicine
Issue Date: 1-Feb-2010
Citation: International Archives of Allergy and Immunology. Vol.151, No.3 (2010), 190-198
Abstract: Background: The house dust mite allergen Der p 2 is one of the most important indoor allergens associated with allergic disease. Recombinant Der (rDer) p 2 with high IgE binding activity can be readily produced in Escherichia coli and Pichia pastoris, but the structure and IgE binding of the different methods of preparation have not been compared. Methods: Secondary structure was assessed by circular dichroism (CD). Intrinsic fluorescence and hydrophobic probe (1-anilinonaphthalene 8-sulphonic acid, ANS) were used to study the Der p 2 hydrophobic cavity. IgE binding was assessed by ELISA inhibition. Results: CD analysis showed the expected secondary structure for both nDer p 2 and refolded Der p 2 prepared from E. coli inclusion bodies but primarily random structure for Der p 2 secreted from P. pastoris. The secreted product, however, had disulphide bonding and could be refolded to a similar structure to natural Der (nDer) p 2 after precipitation with trichloro-acetic or ammonium sulphate. ANS binding and intrinsic Trp92 fluorescence showed that all recombinant proteins were different to nDer p 2 and that the allergen secreted from P. pastoris did not form a hydrophobic cavity. Despite the marked structural changes, all preparations of Der p 2 had similar IgE binding to nDer p 2. Conclusion: Despite almost identical IgE binding, rDer p 2 prepared from both E. coli and P. pastoris showed structural differences to nDer p 2. Der p 2 secreted from P. pastoris lacked most of the natural structure, but refolding could induce the natural structure. Copyright © 2009 S. Karger AG.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=70349422696&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/29275
ISSN: 10182438
Appears in Collections:Scopus 2006-2010

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