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Bicistronic mRNAs to enhance membrane protein overexpression


Marino, Jacopo; Hohl, Michael; Seeger, Markus A; Zerbe, Oliver; Geertsma, Eric (2015). Bicistronic mRNAs to enhance membrane protein overexpression. Journal of Molecular Biology, 427:943-953.

Abstract

Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in E. coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.

Abstract

Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in E. coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

07 Faculty of Science > Department of Chemistry
04 Faculty of Medicine > Institute of Medical Microbiology
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2015
Deposited On:26 Feb 2015 08:00
Last Modified:18 Aug 2018 20:52
Publisher:Elsevier
ISSN:0022-2836
OA Status:Green
Publisher DOI:https://doi.org/10.1016/j.jmb.2014.11.002
PubMed ID:25451035
Project Information:
  • : FunderSNSF
  • : Grant ID310030_141074
  • : Project TitleNMR approaches to study folding and structures of GPCRs

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