The Role of the T2a Peptide in Coexpression of Multiple Genes

The t2a peptide (Two-Amino Acid Peptide) is a viral oligopeptide that can induce ribosomal skipping during translation. It is a self-cleaving peptide that consists of an 18–22 amino acid sequence of the form Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro, which is conserved across different picornaviruses. It has been found to facilitate coexpression of two genes via ribosomal skipping by the formation of a glycyl-proline bond at the 2A/2B junction of the ribosome and therefore results in the production of two proteins with the same N terminus. This phenomenon is referred to as “2A-mediated self-cleavage”.

The first generation of 2A peptides consists of four distinct sequences: F2A (foot and mouth disease virus), E2A (Equine Rhinitis A Virus), P2A (porcine teschovirus-1 2A) and T2A (thosea asigna virus 2A) that are derived from different viruses. These sequences have been shown to exhibit different cleavage efficiency in vivo.

While several studies have shown the advantages of using 2A peptides for the coexpression of multiple genes, their widespread adoption has been limited by two factors. First, it is not clear which 2A sequences exhibit the highest cleavage efficiency in a bi-cistronic setting. Second, there is no widely-adopted cloning vector that harbors a ubiquitous promoter and a 2A gene flanked by multiple cloning sites to enable polycistronic gene expression in cellular contexts.

We compared the cleavage efficiency of the different 2A sequences in a bi-cistronic system and demonstrated that P2A displayed the highest efficiency followed by T2A and E2A. We also tested the effect of varying the preceding N-terminal amino acid sequence, and found that the presence of a hydrophilic glutamate significantly enhanced KLF4 expression compared to that of a non-hydrophilic serine or a positively charged lysine.


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