Nuclear magnetic resonance (NMR) spectroscopy can be used to determine the structure, dynamics and interactions of proteins. However, protein NMR requires stable isotope labelling for signal detection. The cells used for the production of recombinant proteins must therefore be grown in medium containing isotopically labelled substrates. Stable isotope labelling is well established in Escherichia coli, but bacteria are only suitable for the production of simple proteins without post-translational modifications. More complex proteins require eukaryotic production hosts, but their growth can be impaired by labelled media, thus reducing product yields and increasing costs. To address this limitation, we used media supplemented with isotope-labelled substrates to cultivate the tobacco-derived cell line BY-2, which was then cast into plant cell packs (PCPs) for the transient expression of a labelled version of the model protein GB1. Mass spectrometry confirmed the feasibility of isotope labelling with ¹⁵N and ²H using this approach. The resulting NMR spectrum featured a signal dispersion comparable to recombinant GB1 produced in E. coli. PCPs therefore offer a rapid and cost-efficient alternative for the production of isotope-labelled proteins for NMR analysis, especially suitable for complex proteins that cannot be produced in microbial systems.

中文翻译：

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植物细胞包中重组蛋白的瞬时表达有助于核磁共振光谱的稳定同位素标记

核磁共振 (NMR) 光谱可用于确定蛋白质的结构、动力学和相互作用。然而，蛋白质 NMR 需要稳定同位素标记来进行信号检测。因此，用于生产重组蛋白的细胞必须在含有同位素标记底物的培养基中生长。稳定同位素标记在大肠杆菌中得到了很好的建立，但细菌只适合生产没有翻译后修饰的简单蛋白质。更复杂的蛋白质需要真核生产宿主，但它们的生长可能会受到标记培养基的影响，从而降低产品产量并增加成本。为了解决这一限制，我们使用补充了同位素标记底物的培养基来培养烟草衍生细胞系 BY-2，然后将其浇注到植物细胞包 (PCP) 中，以瞬时表达模型蛋白 GB1 的标记版本. 质谱证实了使用这种方法用¹⁵ N 和² H 进行同位素标记的可行性。得到的 NMR 光谱具有与大肠杆菌中产生的重组 GB1 相当的信号分散性. 因此，PCP 为生产用于 NMR 分析的同位素标记蛋白质提供了一种快速且具有成本效益的替代方案，特别适用于无法在微生物系统中生产的复杂蛋白质。