使用工程NEDD8连接酶直接标记小分子蛋白目标
Zachary B Hill1, Samuel B Pollock1, Min Zhuang1
1Department of Pharmaceutical Chemistry and ‡Department of Cellular and Molecular Pharmacology, University of California , San Francisco, California 94158, United States.
Journal of the American Chemical Society
|September 15, 2016
概括
研究人员开发了一种新的近距离标记方法来识别小分子的蛋白质标. 这种技术使用工程酶来标记与特定小分子结合的蛋白质,有助于药物发现和化学探针开发.
科学领域:
- 生物化学
- 化学生物学
- 分子生物学
背景情况:
- 鉴定生物活性小分子的蛋白质标对于药物发现和开发化学探测器至关重要.
- 现有的方法如基于活动的探测器和照片交叉链接器在目标识别方面存在局限性.
- 直接标记那些结合小分子的蛋白质提供了一个有前途的替代方法.
研究的目的:
- 开发一种直接贴近蛋白质的新方法,
- 设计一种用于对小分子结合蛋白的共价标签的杂交链酶.
- 验证该方法在鉴定内源蛋白结合物的有效性.
主要方法:
- 在NEDD8结合酶 (Ubc12) 基础上设计了一种杂交酶.
- 将工程酶与感兴趣的小分子进行共价连接.
- 使用生物化NEDD8衍生物对目标蛋白进行标记.
- 将该方法应用于达沙替尼和其他蛋白质 - 配体相互作用.
主要成果:
- 在复杂细胞溶解物中已知内源蛋白结合物的达沙替尼指导的NEDDylation.
- 通过工程结合酶的合理变异提高了NEDDylation的效率.
- 成功地对酶和另外两种蛋白质 - 配体相互作用进行了亲和导向的NEDDylation.
- 展示了该方法在小分子结合时直接标记蛋白质的能力.
结论:
- 开发了一种有效的近距离标记方法来识别小分子目标.
- 工程联酶和NEDDylation方法提供了直接和正交的策略.
- 这种技术通过识别蛋白质点,便于发现新的化学探针和治疗方法.
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