Kigawa Takanori, Yamaguchi-Nunokawa Emi, Kodama Koichiro, Matsuda Takayoshi, Yabuki Takashi, Matsuda Natsuko, Ishitani Ryuichiro, Nureki Osamu, Yokoyama Shigeyuki
RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan.
J Struct Funct Genomics. 2002;2(1):29-35. doi: 10.1023/a:1013203532303.
Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.
多波长反常衍射相位法对于高通量结构测定特别有用。硒代甲硫氨酸取代的蛋白质通常用于此目的。然而，硒代甲硫氨酸的细胞毒性极大地降低了其在体内表达系统中的掺入效率。在本研究中，使用改进的大肠杆菌无细胞蛋白质合成系统将硒代甲硫氨酸掺入蛋白质中，从而实现高效掺入。通过透析的无细胞系统获得了毫克量的含硒代甲硫氨酸的Ras。质谱分析表明，超过95%的甲硫氨酸残基被硒代甲硫氨酸取代。该蛋白质的晶体在相同条件下生长，并且具有与天然Ras蛋白相同的晶胞常数。通过多波长反常衍射相位法确定的该蛋白质的三维结构与通过体内表达制备的Ras蛋白几乎相同。因此，无细胞合成系统可能成为通过X射线晶体学进行高通量结构测定的强大蛋白质表达方法。
