Abstract
BACKGROUND: Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPON(TM) technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPON(TM) tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions. RESULTS: In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPON(TM)-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆G(total)). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆G(total) tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPON(TM) tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPON(TM) tag increased by 6 kcal/mol. Surprisingly, this new T7AC(rare) variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions. CONCLUSIONS: This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔG(total)), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔG(total). Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPON(TM) tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7AC(rare) tag in biopharmaceutical industry using the CASPON(TM) platform process.