Key result
Synonymous arginine codon mutations improve soluble TEV protease production while tRNA augmentation reduces enzymatic activity.
Why the study?
The effects of synonymous rare arginine codons and tRNA abundance on protein production and activity of TEV protease variants were not fully understood.
Engineering highly soluble TEV protease variants can be achieved by combining amino acid mutations and optimizing specific rare codons, whereas simply augmenting rare tRNA abundance may reduce activity.
May enhance recombinant enzyme yields via codon tuning; leaves open translation to other proteins or clinical production.
It has been identified that a TEV protease (TEVp) variant, TEVp(5M), displays improved solubility. Here, we constructed fifteen TEVp(5M) variants with one or more of six rare arginine codons in the coding sequence replaced with abundant E. coli arginine codons. These codon variants expressed in either E. coli BL21 (DE3) or Rossetta (DE3) showed different solubility and activity. Supply of rare tRNAs increased the tendency of certain codon variants to form insoluble aggregates at early induction stage, as determined by the fused S-tag. About 32% increase in soluble protein production of M5 variant with four synonymously mutated arginine codons was identified in Rossetta (DE3) cells using GFP fusion reporter, comparable to that of TEVp(5M). After purification, two other codon variants from both E. coli strains exhibited less activity than TEVp(5M) on cleaving the native or modified recognition sequence incorporated between GST and E. coli diaminopropionate ammonialyase by enzyme-coupled assay, whereas purified M5 variant showed activity similar to the TEVp(5M). Supply of rare tRNAs caused the decrease of activity of TEVp(5M) and M5 by about 21%. Our results revealed that engineering of highly soluble TEVp variants can be achieved by the combined mutations of amino acid residues and optimization of specific rare codons, whereas simple augment of rare tRNAs abundance resulted in partial loss of activity.
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Fang et al. (2014) studied this question. Synonymous rare arginine codon mutations and rare tRNA supply vs. TEVp 5M (original variant) was evaluated on Soluble protein production and cleavage activity. Synonymous mutations of rare arginine codons improved the soluble production of TEV protease variants, whereas simple augmentation of rare tRNAs resulted in partial loss of enzymatic activity.
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