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

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.

Analysis of 11,430 recombinant protein production experiments reveals that protein yield is tunable by synonymous codon changes of translation initiation sites

“…A recent study also showed that an increase in accessibility of a 30 bp region from the Shine-Dalgarno sequence enhances the expression level of human voltage dependent anion channel in an E. coli cell-free system, which further supports our findings [68]. Overall, the findings show that optimising accessibility is useful for tuning protein expression in both cellular and cell-free expression systems.…”

“…Users can then redirect any selected region to TIsigner for accessibility optimisation. In contrast to the existing gene optimisers, their features are very limited [68,[86][87][88].…”

Protein yield is tunable by synonymous codon changes of translation initiation sites

“…We have implemented our findings in TIsigner web server, which currently supports recombinant protein expression in E. coli and S. cerevisiae (optimisation regions −24:24 and −7:89, respectively; see Fig 1). An independent yet similar implementation is available in XenoExpressO web server with the purpose of optimising protein expression for an E. coli cell-free system ( 59 ). The authors showed that an increase in accessibility of a 30 bp region from the Shine-Dalgarno sequence enhances the expression level of human voltage dependent anion channel, which further supports our findings.…”