A Gel-Encapsulated Bioreactor System for NMR Studies of Protein-Protein Interactions in Living Mammalian Cells
A Gel-Encapsulated Bioreactor System for NMR Studies of Protein-Protein Interactions in Living Mammalian Cells
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DOI：
10.1002/anie.201207243
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Shimada, Ichio
中科院分区：
文献类型：
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作者：
Kubo, Satoshi;Nishida, Noritaka;Shimada, Ichio
细胞内核磁共振波谱是一种用于观察活细胞内同位素标记分子的方法第一次细胞内核磁共振实验是用过表达15n标记蛋白的大肠杆菌进行的首次将细胞内核磁共振技术应用于真核细胞，将同位素标记的靶蛋白微量注射到非洲爪蟾卵母细胞中。[3,4]最近，Inomata等人报道了一种利用细胞穿透标签[5]的新型细胞内核磁共振方法，为细胞内核磁共振波谱在哺乳动物细胞中的应用开辟了道路。本课题组还报道了哺乳动物细胞的细胞内核磁共振方法；我们使用了一种成孔毒素，溶链素O (SLO)，通过扩散引入目标蛋白通过使用这些方法，成功地检测了体内蛋白质-药物相互作用和细胞内翻译后修饰，如磷酸化和乙酰化。[5-7]然而，细胞内核磁共振实验的主要限制是核磁共振测量过程中细胞死亡的发生。由于悬浮液中含有高密度的细胞（> 1 107 mL），在核磁共振管内的厌氧环境中，营养物质的消耗会迅速发生，从而导致条件恶化，导致核磁共振测量期间细胞死亡。[8,9]因此，对活细胞中蛋白质的核磁共振信号的观察受到来自细胞泄漏的同位素标记蛋白质的尖锐核磁共振信号的阻碍因此，真核细胞的细胞内核磁共振实验目前的应用有限，例如在很短的时间内获得单个核磁共振光谱。尽管稀疏采样方法已被用于缩短获得多维核磁共振光谱所需的时间，但许多现有的体外核磁共振实验用于提供有关动力学和蛋白质相互作用的信息，需要几个小时才能完成。为了在更长时间内抑制核磁共振测量期间的细胞死亡，我们利用生物反应器将细胞灌注到核磁共振样管中在早期体内核磁共振研究中，将灌注后的细胞包裹在琼脂糖凝胶中，置于10 mm核磁共振管中，将其隔离在信号检测线圈中，观察酵母和哺乳动物细胞内ATP代谢产物的31P信号，持续数天。[12,13]在这项研究中，我们开发了一种用于细胞内核磁共振波谱的生物反应器（图1）；在这个
In-cell NMR spectroscopy is a method used to observe isotopically labeled molecules within living cells.[1] The first in-cell NMR experiment was performed with an E. coli overexpressing a 15N-labeled protein.[2] For the first application of the in-cell NMR method with eukaryotic cells, isotopically labeled target proteins were introduced, by microinjection, into Xenopus laevis oocytes.[3, 4] Recently, Inomata et al. reported a novel in-cell NMR method utilizing a cell-penetrating tag,[5] which opens the way for the application of in-cell NMR spectroscopy to mammalian cells. Our group also reported an in-cell NMR method for mammalian cells; we used a pore-forming toxin, streptolysin O (SLO), to introduce target proteins by diffusion.[6] By using these methods, protein–drug interactions and intracellular post-translational modifications, such as phosphorylation and acetylation, were successfully detected in vivo.[5–7] However, the major limitation of the in-cell NMR experiments is the occurrence of cell death during the NMR measurement. As the suspension contains a high density of cells (> 1 107 mL), nutrient depletion occurs rapidly in the anaerobic environment within the NMR tube, thus causing the deterioration of conditions and resulting in cell death during NMR measurements.[8, 9] Therefore, the observation of the NMR signals from proteins in living cells is hampered by the sharp NMR signals derived from the isotopically labeled proteins leaked from the cells.[10] Therefore, in-cell NMR experiments for eukaryotic cells currently have limited applications, such as for obtaining a single NMR spectrum measured within a very short time. Although sparse sampling methods have been utilized to shorten the time required to acquire multidimensional NMR spectra,[11] many existing in vitro NMR experiments that are used to provide information regarding dynamics and protein interactions take several hours to perform.To suppress the cell death during NMR measurements over a longer period of time, we utilized a bioreactor to perfuse the cells in the NMR sample tube.[18] In the early in vivo NMR studies, the perfused cells were entrapped in an agarose gel in a 10 mm NMR tube to sequester them in the signal detection coil, and the 31P signals of intracellular ATP metabolites in yeast and mammalian cells were observed for a period of several days.[12, 13] In this study, we developed a bioreactor for in-cell NMR spectroscopy (Figure 1); in this