Influence of the mRNA initial region on protein production: a case study using recombinant detoxified pneumolysin as a model.

Recombinant proteins are of great importance in modern society, mostly as biopharmaceutical products. However, challenging and complex processes with low production yield are major drawbacks. Normally, the optimization to overcome these obstacles is focused on bioreactor and purification processes, and the biomolecular aspects are neglected, seen as less important. In this work, we present how the 5' mRNA secondary structure region can be relevant for translation and, therefore, protein production. For this, BL21(DE3) clones, producing recombinant detoxified pneumolysin (PdT) with and without the N-terminal His-tag, were cultivated in 10-L bioreactors. Another version of the gene (version 2) with synonymous changes in the 5'-end nucleotide sequence was also obtained. Protein production, plasmid stability, carbon sources, and acetic acid were quantified during the cultures. Furthermore, mRNA analyses were performed using TIsigner and RNAfold. The results showed that the His-tag presence at the N-terminus generated a minimum 1.5-fold increase in target protein synthesis, which was explained by the mRNA analyses that returned an mRNA secondary structure easier to translate and, therefore, higher protein production than without the His-tag. The gene version 2 showed lower 5' mRNA opening energy than version 1, allowing higher PdT production even without a tag. This work reveals that simple mRNA analyses during heterologous gene design and production steps can help reach high-recombinant protein titers in a shorter time than using only traditional bioprocess optimization strategies.