Document Type
Article
Publication Date
2019
DOI
10.1038/s41598-019-39382-0
Publication Title
Scientific Reports
Volume
9
Issue
1
Pages
1-15
Abstract
Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.
Original Publication Citation
Mathieu, K., Javed, W., Vallet, S., Lesterlin, C., Candusso, M. P., Ding, F., . . . Orelle, C. (2019). Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain. Scientific Reports, 9(1), 1-15. doi:10.1038/s41598-019-39382-0
ORCID
0000-0002-7470-1948 (Xu, Nancy)
Repository Citation
Mathieu, Khadija; Javed, Waqas; Vallet, Sylvain; Lesterlin, Christian; Candusso, Marie-Pierre; Ding, Feng; Nancy Xu, Xiaohong; Ebel, Christine; Jault, Jean-Michel; and Orelle, Cédric, "Functionality of Membrane Proteins Overexpressed and Purified from E. Coli is Highly Dependent Upon the Strain" (2019). Chemistry & Biochemistry Faculty Publications. 166.
https://digitalcommons.odu.edu/chemistry_fac_pubs/166
Comments
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