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在人类细胞系中对内源蛋白质进行高效标记,以通过单颗粒冷EM进行结构研究

Wooyoung Choi¹, Hao Wu¹, Klaus Yserentant²

¹Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.

Proceedings of the National Academy of Sciences of the United States of America

|July 24, 2023

概括

这项研究介绍了一种CRISPR/Cas9方法,用于有效地标记人类细胞中的蛋白质. 这一进步使得蛋白质在它们的原生环境和细胞过程中进行了详细的结构研究.

科学领域:

分子生物学分子生物学
基因组学就是基因组学.
结构生物学结构生物学

背景情况:

CRISPR/Cas9基因组工程为生物操纵提供了强大的工具,特别是在复杂的生物体中.
研究内源性蛋白质在其原生细胞环境中的研究对于理解生物功能至关重要.

研究的目的:

使用CRISPR/Cas9.9开发一种高效的方法来标记人类细胞系内源性蛋白质.
为了使本地蛋白质的结构研究使用冷电子显微镜.
研究细胞过程中的蛋白质行为以及对刺激的反应.

主要方法:

针对蛋白质标记的同质导向修复供体模板的设计.
编辑基因组的人类细胞系的过渡转染和选择 (HEK293T,Jurkat).
单粒子冷电子显微镜用于结构分析的应用.
在氧化应激下对甘甲3-酸盐脱酶 (GAPDH) 的时空分析.

主要成果:

在HEK293T和Jurkat细胞中成功标记了六种不同的人类蛋白质.
在标记的内源蛋白质上进行结构研究的可行性.
在氧化应激期间观察到GAPDH局部和活动的空间和时间变化.
在GAPDH中与核转位相关的非活性催化位点的增加.

结论:

开发的基于CRISPR/Cas9的策略有助于在人体细胞中有效地标记内源蛋白质.
这种方法增强了对原生蛋白质结构和动态的研究.
这些发现提供了关于GAPDH调节和细胞对氧化应激反应的见解.

关键词:

这就是CRISPR标记.低温电磁波冷却器 (Cryo-EM) 是一个非常好的方法.这是一种内源性蛋白质.人类细胞系的人类细胞系结构的结构结构的结构.

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