Kohno Toshiyuki
Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan.
Methods Mol Biol. 2010;607:113-26. doi: 10.1007/978-1-60327-331-2_11.
Nuclear magnetic resonance (NMR) methods are widely used to determine the three-dimensional structures of proteins, to estimate protein folding, and to discover high-affinity ligands for proteins. However, one of the problems to apply such NMR methods to proteins is that we should obtain mg quantities of (15)N and/or (13)C labeled pure proteins of interest. Here, we describe the method to produce dual amino acid-selective (13)C-(15)N labeled proteins for NMR study using the improved wheat germ cell-free system, which enables sequence-specific assignments of amide signals simply even for very large protein.
核磁共振（NMR）方法被广泛用于确定蛋白质的三维结构、评估蛋白质折叠以及发现蛋白质的高亲和力配体。然而，将此类NMR方法应用于蛋白质时存在的一个问题是，我们需要获得毫克量的感兴趣的（15）N和/或（13）C标记的纯蛋白质。在此，我们描述了一种使用改进的无细胞小麦胚系统生产用于NMR研究的双氨基酸选择性（13）C-（15）N标记蛋白质的方法，该方法即使对于非常大的蛋白质也能简单地实现酰胺信号的序列特异性归属。
