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

利用无细胞翻译系统生产硒代蛋氨酸标记蛋白并应用于X射线晶体结构分析。

• 批准号: 13680692
• 负责人: HORI Hiroyuki
• 金额: $ 2.05万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680692/
• 关键词: Cell-free translation; MAD; selenomethionine; X-ray crystal structure analysis; RNA modification enzymes; 翻訳; 多波長異常分散法; セレノメチオニン

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.

研究了硒代蛋氨酸（SeMet）在小麦胚体外无细胞翻译系统中对两种模型蛋白（GFP和DHFR）的标记。所产生的蛋白质的产率等于通常的蛋氨酸（Met）蛋白质的产率。用LC/MS分析了蛋白质中硒蛋氨酸和蛋氨酸的含量。我们彻底分析了LC色谱图，然而，我们不能检测到来自内在Met的Met标记的肽片段。这些结果有力地表明，foe标记的效率接近100%。在-80℃下保存6个月后，模型蛋白中的SeMet残基没有被氧化，虽然SeMet-GFP的发色团形成比Met-GFP慢，但其荧光光谱的线形与Met-GFP一致，表明SeMet标记没有影响发色团周围的环境。在DHFR的情况下，Met残基包括在M20环中，其在催化循环中起关键作用。我们利用这种标记技术对tRNA甲基转移酶进行了X射线晶体分析。现在，我们将在1.5 μ m分辨率下对晶体进行结构细化。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。

项目成果

H. Takeda, H. Hori, and Y. Endo: "Identification of Aquifex aeolicus tRNA (m^2_2G26) Methyltransferase Gene"Nucleic Acids Res.. Supplement 2. 229-230 (2002)

H. Takeda、H. Hori 和 Y. Endo：“Aquifex aeolicus tRNA (m^2_2G26) 甲基转移酶基因的鉴定”核酸研究补充 2. 229-230 (2002)

J.-M.Park, T.Higuchi, K.Kikuchi, Y.Urano, H.Hori, T.Nishino, J.Aoki, K.Inoue, T.Nagano: "Selective Inhibition of Human Inducible Nitric Oxide Synthase by S-Alkyl-L-Isothiocitrulline-Containing Dipeptide"British J. Pharmachol.. 132. 1876-1882 (2001)

J.-M.Park、T.Higuchi、K.Kikuchi、Y.Urano、H.Hori、T.Nishino、J.Aoki、K.Inoue、T.Nagano：“S 选择性抑制人类诱导型一氧化氮合酶

H. Hori, T. Suzuki, K. Sugawara, Y. Inoue, T. Shibata, S. Kuramitsu, S. Yokoyama, T. Oshima, and K. Watanabe: "Identification and Characterization of tRNA-(Gm18)-methyltransferase from Thermus therm ophilus HB8 ; Domain Structure and Conserved Amino Acid

H. Hori、T. Suzuki、K. Sukawara、Y. Inoue、T. Shibata、S. Kuramitsu、S. Yokoyama、T. Oshima 和 K. Watanabe：“tRNA-(Gm18)-甲基转移酶的鉴定和表征

Kazunori Watanabe, Hiroyuki Hori, Yaeta Endo: "Identification of essential amlnoacid residues of tRNA (Gm18) methyl-transferase for methyl-transfer activity"Nucleic Acids Research Supplement. 1. 33-34 (2001)

Kazunori Watanabe、Hiroyuki Hori、Yaeta Endo：“鉴定用于甲基转移活性的 tRNA (Gm18) 甲基转移酶的必需氨基酸残基”核酸研究增刊。

H.Takeda, H.Hori, Y.Endo: "Identification of A quifex aeclicus tRNA (m^2_2G26) Methyltransferase Gene"Nucleic Acids Res. Supplement. 2. 229-230 (2002)

H.Takeda、H.Hori、Y.Endo：“A quifex aeclicus tRNA (m^2_2G26) 甲基转移酶基因的鉴定”核酸研究。