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Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant.

ABSTRACT: 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.

SUBMITTER: Fang J

PROVIDER: S-EPMC4245098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Synonymous rare codons are considered to be sub-optimal for gene expression because they are translated more slowly than common codons. Yet surprisingly, many protein coding sequences include large clusters of synonymous rare codons. Rare codons at the 5' terminus of coding sequences have been shown to increase translational efficiency. Although a general functional role for synonymous rare codons farther within coding sequences has not yet been established, several recent reports have identified rare-to-common synonymous codon substitutions that impair folding of the encoded protein. Here we test the hypothesis that although the usage frequencies of synonymous codons change from organism to organism, codon rarity will be conserved at specific positions in a set of homologous coding sequences, for example to tune translation rate without altering a protein sequence. Such conservation of rarity-rather than specific codon identity-could coordinate co-translational folding of the encoded protein. We demonstrate that many rare codon cluster positions are indeed conserved within homologous coding sequences across diverse eukaryotic, bacterial, and archaeal species, suggesting they result from positive selection and have a functional role. Most conserved rare codon clusters occur within rather than between conserved protein domains, challenging the view that their primary function is to facilitate co-translational folding after synthesis of an autonomous structural unit. Instead, many conserved rare codon clusters separate smaller protein structural motifs within structural domains. These smaller motifs typically fold faster than an entire domain, on a time scale more consistent with translation rate modulation by synonymous codon usage. While proteins with conserved rare codon clusters are structurally and functionally diverse, they are enriched in functions associated with organism growth and development, suggesting an important role for synonymous codon usage in organism physiology. The identification of conserved rare codon clusters advances our understanding of distinct, functional roles for otherwise synonymous codons and enables experimental testing of the impact of synonymous codon usage on the production of functional proteins.

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Synonymous rare arginine codons and tRNA abundance affect protein production and quality of TEV protease variant.

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