Nuclear magnetic resonance (NMR) spectroscopy is a primary structural biology method to characterize protein dynamics in solution. For large macromolecular systems, methyl-labeling in a perdeuterated background significantly improves the relaxation properties, while providing sensitive probes for structure and dynamics analysis. However, how to prepare methyl-labeled proteins, especially for functional eukaryotic proteins, remains to be a major bottleneck in this field. Due to its advantages in eukaryotic co-translational and post-translational modification, as well as high-density fermentation, Pichia pastoris has been a cost-effective platform strain for 13C, 15N-labeling and deuterium labeling since the early 2000's. Recently, some substantial progress has been made in methyl-labeling, such as the feasibility of 13C isoleucine δ1 methyl-labeling in perdeuterated background and the increased uptake of the Val/Leu precursor. Here, we systematically introduce the isotope-labeling strategies in P. pastoris, including strain engineering and detailed fermentation protocols in 13C, 15N-labeling and methyl-labeling, providing instructions and guidance for the future improvement of sample preparation for NMR spectroscopy.

中文翻译：

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巴斯德毕赤酵母中用于NMR光谱的甲基标记策略的最新进展。

核磁共振波谱学是表征溶液中蛋白质动力学的主要结构生物学方法。对于大分子系统，全氘化背景中的甲基标记显着改善了弛豫性能，同时为结构和动力学分析提供了敏感的探针。但是，如何制备甲基标记的蛋白质，特别是功能性真核蛋白质，仍然是该领域的主要瓶颈。由于其在真核共翻译和翻译后修饰以及高密度发酵方面的优势，自2000年代初以来，巴斯德毕赤酵母一直是13C，15N标记和氘标记的经济有效的平台菌株。最近，甲基标记已取得了一些实质性进展，例如在氘化背景下13C异亮氨酸δ1甲基标记的可行性以及Val / Leu前体的摄取增加。在这里，我们系统地介绍巴斯德毕赤酵母的同位素标记策略，包括菌株工程和13C中详细的发酵方案，15N标记和甲基标记，为将来改进NMR光谱样品制备提供指导和指导。