Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Enhancing the Cell-Free Expression of Native Membrane Proteins by in-Silico Optimisation of the Coding Sequence - An Experimental Study of the Human Voltage-Dependent Anion Channel

Sonja Zayni
,
Samar Damiati
ORCID logo ,
Susana Moreno-Flores
,
Fabian Amman
* ORCID logo ,
Ivo Hofacker
ORCID logo ,
Eva Kathrin Ehmoser
* ORCID logo
Version 1 : Received: 6 July 2021 / Approved: 7 July 2021 / Online: 7 July 2021 (10:20:44 CEST)

A peer-reviewed article of this Preprint also exists.

Zayni, S.; Damiati, S.; Moreno-Flores, S.; Amman, F.; Hofacker, I.; Jin, D.; Ehmoser, E.-K. 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. Membranes 2021, 11, 741. Zayni, S.; Damiati, S.; Moreno-Flores, S.; Amman, F.; Hofacker, I.; Jin, D.; Ehmoser, E.-K. 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. Membranes 2021, 11, 741.

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. This scoring function is publicly available as webservice XenoExpressO at rna.tbi.univie.ac.at.

Keywords

cell-free membrane protein expression; translation enhancer; translation initiation; ribosome docking site; sequence design.

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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