RT Journal Article
SR Electronic
T1 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
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 411694
DO 10.1101/411694
A1 Zayni, Sonja
A1 Damiati, Samar
A1 Moreno-Flores, Susana
A1 Amman, Fabian
A1 Hofacker, Ivo
A1 Ehmoser, Eva-Kathrin
YR 2018
UL http://biorxiv.org/content/early/2018/09/07/411694.abstract
AB 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.