Structural analysis of novel type 6 secretion apparatus proteins

新型6型分泌装置蛋白的结构分析

• 批准号: 21770164
• 负责人: KANAMARU Shuji
• 金额: $ 2.16万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Young Scientists (B)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21770164/
• 关键词: グラム陰性菌; 分泌装置; バクテリオファージ; 構造解析; T6SS; βヘリックス; VgrG; グラム陰性菌; 分泌装置; バクテリオファージ; 構造解析; T6SS; βヘリックス; VgrG

To determine the crystal structure of VgrG protein of Type 6 secresion apparatus from E. coli O157 and E. coli CFT073, over-expression vectors for both full-length and C-terminal domain of VgrG were constructed. We succeeded in establish the purification methods for VgrG from E. coli O157. Purified protein was treated with trypsin to get stable VgrG1^M467-his. This VgrG1^M467-his was examined thousands of crystallization condition. One of the conditions gave nice crystal formation for structure determination. Now we are trying get isomorphous heavy atom derivatives for phase determination. Moreover, we are going to establish other VgrG purification strategy.

为了确定来自大肠杆菌O157和大肠杆菌CFT073的6型分级设备的VGRG蛋白的晶体结构，构建了VGRG的全长和C末端结构域的过表达向量。我们成功地建立了大肠杆菌O157的VGRG的纯化方法。用胰蛋白酶处理纯化的蛋白质，以获得稳定的VGRG1^M467-HIS。该VGRG1^M467-HIS被检查了数千种结晶条件。其中一种条件为结构确定提供了很好的晶体形成。现在，我们正在尝试获得同构重原子衍生物以进行相确定。此外，我们将建立其他VGRG纯化策略。