T2A Peptide As a Bi-Cistronic Reporter

t2a peptide

2A self-cleaving peptides are 18–22 aa long sequences that can induce ribosome skipping during translation of a target protein. They are widely used in many eukaryotic viruses as part of a ribosome-pausing strategy. The peptides cleave between the Gly and Pro residues at their C-terminus and are active in ribosomes of all eukaryotic species tested.

t2a peptides are also efficient for polycistronic or bi-cistronic expression of genes from a single mRNA (3-6, 6, 7). They also promote translational recoding through an ‘StopGo’ reaction in which a polypeptide upstream and a peptide downstream of the 2A cleave to form two separate proteins from the same ORF. These two products are re-translated into a new polypeptide sequence at the point of the 2A cleavage, thus mediating the recoding event. Unlike other known ribosome-pausing peptides, this’stop-carry on’ recoding is not dependent on phosphorylation or tRNA binding.

In order to test the activity of 2A peptides, we designed a bi-cistronic reporter system for screening its ability to drive translational recoding. The construct consisted of a constitutively active promoter, an upstream ORF encoding red fluorescent protein fused to a HA tag, a 2A peptide of interest, and a GFP fused to a NLS sequence.

We performed Western blotting and flow cytometry assays on the resulting transcripts. The ratio of fused to separated polypeptides was determined by immunoblotting, and a corresponding copy number was measured using real-time quantitative PCR.

Amino acid substitutions introduced within 30-40 aa upstream of the cleavage site, in the case of short F2A and T2A peptides, can have a profound effect on their cleavage efficiency. The -AAKA- mutation upstream of F2A significantly increased its activity, and the -SGSRGAC- substitution upstream of T2A decreased it. These results show that amino acid substitutions immediately upstream of the cleavage site should be carefully considered in the design of 2A peptide constructs for coexpression of multiple proteins.


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