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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. DOI: https://doi.org/10.1016/j.jmb.2014.11.002 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-108581 Accepted Version Originally published at: 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. DOI: https://doi.org/10.1016/j.jmb.2014.11.002 AC CE PT ED M AN US CR IP T ACCEPTED MANUSCRIPT 1 Bicistronic mRNAs to enhance membrane protein overexpression Jacopo Marino, Michael Hohl, Markus A. Seeger, Oliver Zerbe and Eric R. Geertsma a Department of Chemistry, University of Zurich, Zurich, Switzerland. b Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland. c Institute of Biochemistry, Goethe-University Frankfurt, Frankfurt am Main, Germany * present address: Gene Center, Department of Chemistry and Biochemistry, University of Munich, Germany # corresponding author. E-mail: [email protected]. Institute of Biochemistry, Biocenter N200/1.08, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M., Germany; phone: +49 (0)69-798-29255; fax: +49 (0)69798-29244. AC CE PT ED M AN US CR IP T ACCEPTED MANUSCRIPT