Electron microscopic study of the human MDN1 protein

doi:10.3969/j.issn.1674-8115.2021.05.001

Abstract

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

CLC Number:

Q518.2

Yun-tao XU, Ming-yue LI, Ming LEI. Electron microscopic study of the human MDN1 protein[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(5): 559-564.

Figures/Tables 3

TrendMD

References 32

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