Ippel Johannes H, Pouvreau Laurice, Kroef Toos, Gruppen Harry, Versteeg Geurt, van den Putten Peter, Struik Paul C, van Mierlo Carlo P M
Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands.
Proteomics. 2004 Jan;4(1):226-34. doi: 10.1002/pmic.200300506.
Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.
蛋白质的同位素标记对于结构蛋白质组学领域的进展至关重要。它能够利用核磁共振波谱（NMR）的强大功能来表征蛋白质的三维结构及相应的动力学特征。获得同位素标记蛋白质分子的常用方法是在富含同位素的培养基上生长的细菌或酵母宿主生物体中表达相应基因。这种方法有几个缺点。在此，我们证明了用(^{15}N)同位素完全标记一株植物是可行的。对高等生物进行体内标记的优势在于所有组成蛋白质都被标记，并可作为经过翻译后修饰、正确折叠的功能性蛋白质获得。采用水培装置创建了第一个均匀(^{15}N)标记（>98%）的植物物种实例，即马铃薯植株（Solanum tuberosum L.，品种Elkana）。使用硝酸钾 - ([^{15}N])作为唯一氮源，以低成本种植了两株植物。在收获时，共收集了3.6千克马铃薯块茎以及1.6千克枝叶、匍匐茎和根，所有这些都被完全(^{15}N)标记。从块茎中分离出了克级数量的可溶性(^{15}N)标记蛋白质（主要由糖蛋白马铃薯素和库尼茨型蛋白酶抑制剂组成）。对马铃薯汁液完整蛋白质组以及一种分离出的蛋白酶抑制剂的NMR结果说明了标记过程的成功。所提出的同位素标记方法易于修改以标记其他植物。文中还讨论了其在植物结构蛋白质组学领域预期产生的影响。
