上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2021 , Vol. 41 >Issue 5: 559 - 564

DOI: https://doi.org/10.3969/j.issn.1674-8115.2021.05.001

创新团队成果专栏

人源MDN1蛋白质的电镜结构研究

  • 许云涛 ,
  • 李明月 ,
  • 雷鸣
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  • 上海交通大学医学院附属第九人民医院上海精准医学研究院，上海 200125
许云涛（1995—），男，硕士生；电子信箱：xyt347590681@sjtu.edu.cn。

网络出版日期: 2021-05-27

基金资助

国家自然科学基金(31525007)

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Electron microscopic study of the human MDN1 protein

  • Yun-tao XU ,
  • Ming-yue LI ,
  • Ming LEI
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  • Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China

Online published: 2021-05-27

Supported by

National Natural Science Foundation of China(31525007)

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摘要

目的·利用负染电镜技术分析人源midasin AAA-ATPase 1（MDN1，又称Rea1）蛋白质结构。方法·利用CRISPR/Cas9基因编辑方法在Expi293F细胞内源MDN1蛋白质的氨基端敲入3×FLAG纯化标签，采用ANTI-FLAG? M2 Agarose Affinity Gel亲和层析和甘油密度梯度离心的方法分离纯化目的蛋白质；通过负染色电镜技术和单颗粒（single-particle）重构技术探究人源MDN1蛋白质结构。结果·利用亲和层析及密度梯度离心方法分离纯化获得高纯度、均一性较好的人源MDN1蛋白质样品；采用甲酸铀负染色后利用120 kV电镜初步解析了目的蛋白质MDN1的空间结构。结论·利用单颗粒重构技术搭建了人源MDN1蛋白质的低分辨率的负染模型。

关键词： MDN1蛋白质; pre-60S核糖体; 核糖体成熟; 负染电镜

本文引用格式

许云涛 , 李明月 , 雷鸣 . 人源MDN1蛋白质的电镜结构研究[J]. 上海交通大学学报（医学版）, 2021 , 41(5) : 559 -564 . DOI: 10.3969/j.issn.1674-8115.2021.05.001

Abstract

Objective

·To study the structure of the human midasin AAA-ATPase 1 (MDN1, Rea1) protein by negative-staining electron microscopy.

Methods

·Using the CRISPR/Cas9 genome editing method, a 3×FLAG affinity tag was inserted into the N-terminus of MDN1 in Expi293F cells. Tagged proteins were isolated via affinity purification with ANTI-FLAG? M2 Agarose Affinity Gel, followed by glycerol density gradient centrifugation. The purified protein sample was then subjected to negative-staining electron microscopy and single particle image analysis.

Results

·The FLAG-tagged endogenous MDN1 proteins with high purity and good homogeneity were obtained using affinity chromatography and density gradient centrifugation. Preliminary study on the structure of human MDN1 was achieved by 120 kV electron microscope after negative staining with uranium formate.

Conclusion

·A low resolution model of human MDN1 protein was achieved by single particle reconstruction analysis.

Key words： MDN1 protein; pre-60S ribosome; ribosome maturation; negative-staining electron microscopy

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