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

Recombinant protein production is a key process in generating proteins of interest in the pharmaceutical industry and biomedical research. However, about 50% of recombinant proteins fail to be expressed in a variety of host cells. Here we show that the accessibility of translation initiation sites modelled using the mRNA base-unpairing across the Boltzmann’s ensemble significantly outperforms alternative features. This approach accurately predicts the successes or failures of expression experiments, which utilised Escherichia coli cells to express 11,430 recombinant proteins from over 189 diverse species. On this basis, we develop TIsigner that uses simulated annealing to modify up to the first nine codons of mRNAs with synonymous substitutions. We show that accessibility captures the key propensity beyond the target region (initiation sites in this case), as a modest number of synonymous changes is sufficient to tune the recombinant protein expression levels. We build a stochastic simulation model and show that higher accessibility leads to higher protein production and slower cell growth, supporting the idea of protein cost, where cell growth is constrained by protein circuits during over expression.Author summary: Recombinant proteins are widely used as therapeutics, such as vaccines, monoclonal antibodies, hormones and enzymes. However, the success rate of recombinant protein production is about 50%. To address this problem, we propose optimising the unpairing propensities of nucleotides around translation initiation sites using a thermodynamic quantity called mRNA accessibility. Our study shows that this method is generalisable across prokaryotic and eukaryotic expression hosts. Importantly, we validated this method using laboratory experiments and computational modelling. Furthermore, we propose a low cost technique to tune protein expression by engineering minimal changes to genes of interest through our web application (https://tisigner.com/tisigner).