High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies
High-yield expression and purification of isotopically labeled norcoclaurine synthase, a Bet v 1-homologous enzyme, from Thalictrum flavum for NMR studies
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DOI：
10.1016/j.pep.2007.07.010
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发表时间：
2007-12-01
影响因子：
1.6
通讯作者：
Mateko, Irena
中科院分区：
文献类型：
--
作者：
Berkner, Hanna;Engelhorn, Julia;Mateko, Irena
在常见的草甸芸香、黄菊和其它植物中发现的酶去甲丁香碱合酶（NCS）显示出与致病相关（PR 10）蛋白质的10类成员的序列同源性，所述致病相关（PR 10）蛋白质含有变应原，例如主要的桦树花粉变应原Betv 1、主要的樱桃变应原Pru av 1和主要的苹果变应原Mal d 1。这种酶参与植物的次级代谢，是产生吗啡等生物活性次级代谢产物所必需的。尽管PR 10类过敏原的生理功能仍然未知，但已经详细研究了NCS活性。因此，通过NMR光谱研究NCS的结构特性不仅可以提供有关酶的反应机制的新信息，而且还有望帮助澄清长期存在和激烈争论的生理功能问题以及该蛋白质家族成员致敏潜力的原因。作为三维溶液结构的第一个重要步骤，我们优化了重组NCS在大肠杆菌中的表达，并建立了一个有效的纯化方案，产生大量的纯同位素标记的活性酶。NCS的身份通过电喷雾电离质谱法确认，并且通过检测放射性标记的反应产物的测定来确定纯化的酶的活性。通过NMR光谱的光谱分析表明，蛋白质正确折叠，具有明确的三级结构。(C)2007年爱思唯尔公司All rights reserved.
The enzyme norcoclaurine synthase (NCS) found in the common meadow rue, Thalictrum flavum, and other plants shows sequence homology to members of the class 10 of pathogenesis related (PR 10) proteins that contains allergens such as the major birch pollen allergen Bet v 1, the major cherry allergen Pru av 1, and the major apple allergen Mal d 1. The enzyme is involved in the plant's secondary metabolism and is required for the production of bioactive secondary metabolites like morphine. Whereas the physiological function of PR 10 class allergens is still unknown, NCS activity has been studied in detail. Investigation of the structural properties of NCS by NMR spectroscopy can thus not only provide new information concerning the reaction mechanism of the enzyme, but is also expected to help clarify the long standing and heavily debated question on the physiological function as well as the reasons for the allergenic potential of members of this protein family. As the first important step towards the three-dimensional solution structure, we optimized expression of recombinant NCS in Escherichia coli and established an efficient purification protocol yielding high amounts of pure isotopically labeled active enzyme. The identity of NCS was confirmed by electrospray ionization mass spectrometry, and activity of the purified enzyme was determined by an assay detecting the radiolabeled reaction product. Spectroscopic analysis by NMR spectroscopy showed that the protein was properly folded with well defined tertiary structure. (C) 2007 Elsevier Inc. All rights reserved.