Kigawa T, Muto Y, Yokoyama S
Department of Biophysics and Biochemistry, School of Science, University of Tokyo, Japan.
J Biomol NMR. 1995 Sep;6(2):129-34. doi: 10.1007/BF00211776.
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity of labeling in in vivo expression systems. In the present study, a cell-free protein synthesis system was optimized, so that highly efficient and selective stable isotope labeling of proteins can be achieved in the absence of amino acid metabolism. The productivity of the E. coli cell-free coupled transcription-translation system was first improved, by about fivefold, by using the T7 RNA polymerase for transcription and also by improving the translation conditions. Thus, about 0.1 mg protein per 1 ml reaction mixture was synthesized. Then, this improved cell-free system was used for Asp- or Ser-selective 15N-labeling of the human c-Ha-Ras protein. With a 15 ml cell-free reaction, using less than 1 mg of 15N-labeled amino acid, 1 mg of the Ras protein was obtained. 1H-15N HSQC experiments confirmed that the Ras protein was efficiently labeled with high selectivity. These results indicate that this cell-free protein synthesis system is useful for NMR studies.
对于将多维核磁共振光谱应用于更大的蛋白质而言，对20种氨基酸之一进行选择性标记将会很有用。然而，对于某些氨基酸，氨基酸代谢会大大降低体内表达系统中标记的效率和选择性。在本研究中，对无细胞蛋白质合成系统进行了优化，以便在不存在氨基酸代谢的情况下实现蛋白质的高效和选择性稳定同位素标记。首先通过使用T7 RNA聚合酶进行转录并改善翻译条件，将大肠杆菌无细胞偶联转录-翻译系统的产量提高了约五倍。因此，每1 ml反应混合物合成了约0.1 mg蛋白质。然后，将这种改进的无细胞系统用于人c-Ha-Ras蛋白的天冬氨酸或丝氨酸选择性15N标记。在15 ml无细胞反应中，使用少于1 mg的15N标记氨基酸，获得了1 mg的Ras蛋白。1H-15N HSQC实验证实，Ras蛋白被高效且高选择性地标记。这些结果表明，这种无细胞蛋白质合成系统对核磁共振研究很有用。
