Production of selenomethionine labeled proteins by cell-free translation system and application to X-ray crystal structure analysis.

在体外无细胞翻译系统中研究了硒代蛋氨酸（SeMet）对两种模式蛋白（GFP和DHFR）的标记。所得蛋白质的产率与通常的蛋氨酸（Met）蛋白质的产率相等。采用液相色谱/质谱法分析蛋白质中SeMet和Met的含量。我们彻底分析了LC色谱图，但是我们无法检测到来自内在Met的Met标记肽片段。这些结果强烈表明，敌人标记的效率接近100%。此外，我们还发现模型蛋白中的SeMet残基在-80℃下保存6个月没有被氧化。虽然与Met-GFP相比，SeMet- gfp的发色团形成较慢，但其荧光光谱的线形与Met-GFP一致，说明SeMet标记不影响发色团周围的环境。在DHFR中，Met残留物包含在M20环中，M20环在催化循环中起关键作用。然而，SeMet标记对酶活性没有明显的影响。我们利用这种标记技术应用于tRNA甲基转移酶的x射线晶体分析。现在，我们将在1.5Å分辨率下对晶体进行结构改进。基于这些实验结果，我们证实了无细胞翻译系统适用于结构蛋白质组学。

We investigated the selenomethionine (SeMet) labeling of two model proteins (GFP and DHFR) by wheat germ in vitro cell-free translation system. The yields of the produced proteins were equal to those of the usual methionine (Met) proteins. We analyzed the contents of SeMet and Met in the proteins by LC/MS spectrometry. We analyzed the LC chromatograms thoroughly, however, we could not detect the Met labeled peptide fragment derived from the intrinsic Met. These results strongly suggest that the efficiency of foe labeling was near 100%. Further, we also found that SeMet residues in the model proteins were not oxidized during the store at -80℃ for 6 months.Although the formation of the chromophore of SeMet-GFP was slow as compared to Met-GFP, the line shape of the fluorescence spectrum of the protein coincides with that of Met-GFP, suggesting that the SeMet labeling did not affect the environment around the chromophore. In the case of DHFR, Met residues are included the M20 loop, which plays a key role in the catalytic cycles. However, SeMet labeling did not change the enzymatic activity apparently.We utilized this labeling technique to apply the X-ray crystal analysis of tRNA methyltransferase. Now, we are going on the structural refinement of the crystal at 1.5Å resolutions. Based on these experimental results, we confirmed that cell-free translation system was applicable to the structural proteomics.