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Enhancing the cell-free expression of native membrane proteins by in-silico optimization of the coding sequence – an experimental study of the human voltage-dependent anion channel

Sonja Zayni, View ORCID ProfileSamar Damiati, Susana Moreno-Flores, Fabian Amman, View ORCID ProfileIvo Hofacker, View ORCID ProfileEva-Kathrin Ehmoser
doi: https://doi.org/10.1101/411694
Sonja Zayni
1Department of Nanobiotechnology, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences (BOKU), Muthgasse 11, A-1190, Vienna, Austria
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Samar Damiati
2Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
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Susana Moreno-Flores
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Fabian Amman
4Institute for Theoretical Chemistry – Theoretical Biochemistry Group, University of Vienna, Währinger Straße 17, A-1090 Vienna, Austria
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Ivo Hofacker
4Institute for Theoretical Chemistry – Theoretical Biochemistry Group, University of Vienna, Währinger Straße 17, A-1090 Vienna, Austria
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Eva-Kathrin Ehmoser
1Department of Nanobiotechnology, Institute for Synthetic Bioarchitectures, University of Natural Resources and Life Sciences (BOKU), Muthgasse 11, A-1190, Vienna, Austria
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Abstract

The investigation of membrane proteins, key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage dependent anion channel (VDAC), a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function is proposed that quantifies the feasibility of formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome. The scoring function enables to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted September 07, 2018.
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Enhancing the cell-free expression of native membrane proteins by in-silico optimization of the coding sequence – an experimental study of the human voltage-dependent anion channel
Sonja Zayni, Samar Damiati, Susana Moreno-Flores, Fabian Amman, Ivo Hofacker, Eva-Kathrin Ehmoser
bioRxiv 411694; doi: https://doi.org/10.1101/411694
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Enhancing the cell-free expression of native membrane proteins by in-silico optimization of the coding sequence – an experimental study of the human voltage-dependent anion channel
Sonja Zayni, Samar Damiati, Susana Moreno-Flores, Fabian Amman, Ivo Hofacker, Eva-Kathrin Ehmoser
bioRxiv 411694; doi: https://doi.org/10.1101/411694

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