Abstract
mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.
Highlights
Gene expression is extensively regulated at different levels, including transcription, mRNA degradation, translation and protein degradation [1]
Translational attenuation sites in epoxide hydrolases (EHs) from A. radiobacter influence its solubility in E. coli
E. coli does not encode any EH to assess a translation profile of endogenous enzyme whose expression would be evolutionarily optimized for the E. coli tRNAome
Summary
Gene expression is extensively regulated at different levels, including transcription, mRNA degradation, translation and protein degradation [1]. Growth of the host at lower temperatures, which globally slows down translation, improves the functional expression of proteins [30] by increasing the time window for co-translational folding of each domain [27]. Adaptation of the translation profile of a heterologous gene to the tRNAome of the expression host has more potential to enhance its expression [32] but requires knowledge of the tRNA concentration of both the native and expression strains. We introduced synonymous substitutions to decrease the folding energy of the initial 5’-coding region of one EH which enhanced the expression level by facilitating translation initiation. Folding energy in the initial 5’ coding region are successful strategies to fine-tune the expression of heterologous proteins
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