Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2
Expression and secondary structure determination by NMR methods of the major house dust mite allergen Der p 2
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DOI：
10.1074/jbc.272.43.26893
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发表时间：
1997-10-24
影响因子：
4.8
通讯作者：
Benjamin, DC
中科院分区：
文献类型：
--
作者：
Mueller, GA;Smith, AM;Benjamin, DC
哮喘与室内变应原的致敏作用密切相关。本研究用异核核磁共振法测定了屋尘螨主要变应原Der-p2的二级结构，将其从酵母表达载体pSAY1亚克隆到高产细菌表达载体pET21a中，产量为50 mg/L。用竞争抑制酶联免疫吸附试验(ELISA)和改良的单抗放射变态反应试验(RAST)证明重组蛋白具有与天然尘螨蛋白相当的免疫反应性。通过检测化学位移、短程和长程NOESYs、J(HN-HA-)偶联常数和酰胺交换率，确定了重组蛋白的二级结构。从这些数据可以清楚地看出，Der p 2是由β-折叠和随机卷曲组成的，基于长程距离限制，一些β链被排列成两个三链的、反平行的β-折叠。
There exists a strong correlation between asthma and sensitization to indoor allergens, This study reports on the secondary structure of the major house dust mite allergen Der p 2, determined using heteronuclear NMR methods, The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter, The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST), The secondary structure was determined by examining chemical shifts, short and long range NOESYs, J(HN-HA) coupling constants, and amide exchange rates, From these data, it is clear that Der p 2 is composed of beta-sheets and random coil, Based on long range distance constraints, a number of beta strands were aligned into two three-stranded, antiparallel beta-sheets.