Matsuda Takayoshi, Koshiba Seizo, Tochio Naoya, Seki Eiko, Iwasaki Noriyuki, Yabuki Takashi, Inoue Makoto, Yokoyama Shigeyuki, Kigawa Takanori
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
J Biomol NMR. 2007 Mar;37(3):225-9. doi: 10.1007/s10858-006-9127-5. Epub 2007 Jan 20.
Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium L-glutamate (L-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium D-glutamate (D-Glu system). The productivity of the D-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H-15N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the D-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the D-Glu system. These results indicate that the D-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins.
无细胞蛋白质合成适用于对蛋白质进行稳定同位素标记以用于核磁共振分析。含有乙酸钾用于高效翻译的大肠杆菌无细胞系统（KOAc系统）通常用于稳定同位素标记，尽管其产量低于其他系统。含有高浓度L-谷氨酸钾的系统（L-Glu系统），而非乙酸钾，产量很高，但不能用于Glu残基的稳定同位素标记。在本研究中，我们开发了一种使用D-谷氨酸钾的新型无细胞系统（D-Glu系统）。D-Glu系统的产量约为KOAc系统的两倍。用D-Glu系统制备的均匀稳定同位素标记的Ras蛋白的1H-15N HSQC谱中的交叉峰强度与用KOAc系统获得的相似，只是D-Glu系统产生的蛋白质的Asp强度要高得多。这些结果表明，D-Glu系统是一种高产的无细胞系统，特别适用于蛋白质的稳定同位素标记。
