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

Abstract

149/150 words) Recombinant protein production in microbial systems is well-established, yet half of these experiments have failed in the expression phase. Failures are expected for 'difficult-to-express' proteins, but for others, codon bias, mRNA folding, avoidance, and G+C content have been suggested to explain observed levels of protein expression. However, determining which of these is the strongest predictor is still an active area of research. We used an ensemble average of energy model for RNA to show that the accessibility of translation initiation sites outperforms other features in predicting the outcomes of 11,430 experiments of recombinant protein production in Escherichia coli . We developed TIsigner and showed that synonymous codon changes within the first nine codons are sufficient to improve the accessibility of translation initiation sites. Our software produces scores for both input and optimised sequences, so that success/failure can be predicted and prevented by PCR cloning of optimised sequences.

Features of Functional Human Genes

“…Since functional ncRNAs are also likely to form secondary structures with a lower free energy, we measured the minimum free energy (MFE) structure using either a singular sequence or sequence alignment (Lorenz et al 2011). Accessibility, which is a measure of the availability of nucleotides for base-pairing, has also been shown to be important for mRNA translation rates, and thus could be associated with functionality (Bhandari et al 2019). Both covariation features produce significant correlations for all functionally assigned sequences, with short ncRNA minimum covariance E-value and maximum covariance for all sequence types ranking above the neutral predictor, making the latter highly associated with functionality (Figures 1 & 2).…”

“…R-scape 1.4.0 was used to calculate maximum covariance score and minimum covariance E-value (Rivas et al 2017). RNAfold and RNAplfold from ViennaRNA 2.4.14 were used to calculate both MFE and accessibility values for each region (Lorenz et al 2011; Bhandari et al 2019).…”

Solubility-Weighted Index: fast and accurate prediction of protein solubility

“…We linked SoDoPE with TIsigner, which is our existing web server for maximising the accessibility of translation initiation sites (Bhandari, Lim, and Gardner 2019) . This pipeline allows users to predict and optimise both protein expression and solubility for a gene of interest.…”

Improving recombinant protein production using TISIGNER.com

“…TIsigner offers tunable protein expression by optimising the mRNA accessibility of translation initiation sites (23) . The regions used to calculate accessibility (opening energy) are specific to the expression hosts, which is calculated using RNAplfold (28)(29)(30) .…”

“…However, recent systematic studies suggest mRNA secondary structure (minimum free energy) is the most important feature in protein expression (21,22) . We have recently shown that the mRNA accessibility of translation initiation sites outperforms mRNA secondary structure (minimum free energy) in predicting the expression of 11,430 recombinant proteins (23) .…”