Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O
Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin, Streptolysin O
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DOI：
10.1021/ja904407w
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发表时间：
2009-08-12
影响因子：
15
通讯作者：
Shimada, Ichio
中科院分区：
文献类型：
--
作者：
Ogino, Shinji;Kubo, Satoshi;Shimada, Ichio
我们开发了一种新的细胞内核磁共振方法，适用于任何类型的细胞，不需要靶蛋白修饰或专门的设备。稳定同位素标记的靶蛋白胸腺素β 4 （T β 4）被递送到293F细胞，T β 4被一种成孔毒素溶血素O渗透，并在T β 4被摄取后被Ca2+重新封闭。结果，我们成功地观察到源自T β 4的H-1-N-15 HSQC信号，包括那些来自n端乙酰化的信号，这是在细胞内作为翻译后修饰发生的。
We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta 4 (T beta 4), was delivered to 293F Cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca2+ after T beta 4 uptake. As a result, we successfully observed H-1-N-15 HSQC signals originating from the T beta 4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.