Institute of Molecular Biology and Genetics, Mahidol University, Salaya, Thailand.
Int Arch Allergy Immunol. 2010;151(3):190-8. doi: 10.1159/000242356. Epub 2009 Sep 29.
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.
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.
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.
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.
屋尘螨过敏原 Der p 2 是与过敏疾病相关的最重要的室内过敏原之一。具有高 IgE 结合活性的重组 Der（rDer）p 2 可以在大肠杆菌和毕赤酵母中容易地生产，但不同制备方法的结构和 IgE 结合尚未进行比较。
采用圆二色性（CD）评估二级结构。用固有荧光和疏水性探针（1-苯胺基萘-8-磺酸，ANS）研究 Der p 2 的疏水性腔。通过 ELISA 抑制法评估 IgE 结合。
CD 分析显示，大肠杆菌包涵体中制备的 nDer p 2 和复性 Der p 2 具有预期的二级结构，但毕赤酵母分泌的 Der p 2 主要为无规卷曲结构。然而，分泌产物具有二硫键，并可在三氯乙酸或硫酸铵沉淀后复性为类似于天然 Der（nDer）p 2 的结构。ANS 结合和固有色氨酸 92 荧光表明，所有重组蛋白均与 nDer p 2 不同，并且毕赤酵母分泌的过敏原未形成疏水性腔。尽管结构发生了明显变化，但 Der p 2 的所有制备物与 nDer p 2 的 IgE 结合相似。
尽管具有几乎相同的 IgE 结合，但从大肠杆菌和毕赤酵母制备的 rDer p 2 与 nDer p 2 表现出结构差异。毕赤酵母分泌的 Der p 2 缺乏大部分天然结构，但复性可以诱导天然结构。
