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 overexpression.

TISIGNER.com: web services for improving recombinant protein production.

Experiments that are planned using accurate prediction algorithms will mitigate failures in recombinant protein production. We have developed TISIGNER (https://tisigner.com) with the aim of addressing technical challenges to recombinant protein production. We offer three web services, TIsigner (Translation Initiation coding region designer), SoDoPE (Soluble Domain for Protein Expression) and Razor, which are specialised in synonymous optimisation of recombinant protein expression, solubility and signal peptide analysis, respectively. Importantly, TIsigner, SoDoPE and Razor are linked, which allows users to switch between the tools when optimising genes of interest.

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

MOTIVATION: Recombinant protein production is a widely used technique in the biotechnology and biomedical industries, yet only a quarter of target proteins are soluble and can therefore be purified. RESULTS: We have discovered that global structural flexibility, which can be modeled by normalized B-factors, accurately predicts the solubility of 12 216 recombinant proteins expressed in Escherichia coli. We have optimized these B-factors, and derived a new set of values for solubility scoring that further improves prediction accuracy. We call this new predictor the 'Solubility-Weighted Index' (SWI). Importantly, SWI outperforms many existing protein solubility prediction tools. Furthermore, we have developed 'SoDoPE' (Soluble Domain for Protein Expression), a web interface that allows users to choose a protein region of interest for predicting and maximizing both protein expression and solubility. AVAILABILITY AND IMPLEMENTATION: The SoDoPE web server and source code are freely available at https://tisigner.com/sodope and https://github.com/Gardner-BinfLab/TISIGNER-ReactJS, respectively. The code and data for reproducing our analysis can be found at https://github.com/Gardner-BinfLab/SoDoPE_paper_2020. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.