Ayoub, Nooraldeen; Roth, Patrick; Ucurum, Zöhre; Fotiadis, Dimitrios; Hirschi, Stephan (2023). Structural and biochemical insights into His-tag-induced higher-order oligomerization of membrane proteins by cryo-EM and size exclusion chromatography. Journal of structural biology, 215(1), p. 107924. Elsevier 10.1016/j.jsb.2022.107924
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Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.
Item Type: |
Journal Article (Original Article) |
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Division/Institute: |
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine |
Graduate School: |
Graduate School for Cellular and Biomedical Sciences (GCB) |
UniBE Contributor: |
Ayoub, Nooraldeen Fathi Nooraldeen, Roth, Patrick, Ucurum Fotiadis, Zöhre, Fotiadis, Dimitrios José, Hirschi, Stephan |
Subjects: |
500 Science > 570 Life sciences; biology 600 Technology > 610 Medicine & health |
ISSN: |
1047-8477 |
Publisher: |
Elsevier |
Language: |
English |
Submitter: |
Pubmed Import |
Date Deposited: |
05 Dec 2022 13:41 |
Last Modified: |
01 Dec 2023 00:25 |
Publisher DOI: |
10.1016/j.jsb.2022.107924 |
PubMed ID: |
36462717 |
Uncontrolled Keywords: |
His-tag cryo-EM membrane protein oligomerization protein aggregation size exclusion chromatography |
BORIS DOI: |
10.48350/175470 |
URI: |
https://boris.unibe.ch/id/eprint/175470 |
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