High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice

Kim et al. (2011). PLoS ONE DOI: 10.1371/journal.pone.0018556 Citations: 1465

2A peptides could replace bulky IRES elements, but their cleavage efficiencies were untested in vivo. F2A, E2A, T2A, and P2A were compared in human cells, zebrafish, and mouse liver. P2A from porcine teschovirus-1 gave the highest cleavage efficiency in every context.

Parts used

P2A Self-Cleaving Peptide T2A Self-Cleaving Peptide E2A Self-Cleaving Peptide F2A Self-Cleaving Peptide EGFP mCherry

Key findings

The researchers compared four viral 2A peptides (F2A from foot-and-mouth disease virus, E2A from equine rhinitis A virus, T2A from Thosea asigna virus, and P2A from porcine teschovirus-1) for cleavage efficiency using Western blotting and confocal microscopy. P2A demonstrated the highest cleavage efficiency across all tested systems, followed by T2A, E2A, and F2A. The efficiency was quantified by measuring separation of NLS-EGFP and mCherry-CAAX fusion proteins into distinct nuclear and membrane localizations.
Validation studies in HEK293T, HT1080, and HeLa human cell lines showed P2A achieved superior protein separation compared to other 2A variants, with cleaved products correctly localized to nucleus (NLS-EGFP) and plasma membrane (mCherry-CAAX) as detected by confocal microscopy at 24 hours post-transfection. The cleavage efficiency pattern was recapitulated in zebrafish embryos at 24 hours post-fertilization and mouse liver tissue at 3 days post-injection with recombinant adenovirus.
Expression plasmids and adenoviral shuttle vectors containing each 2A sequence were generated using a pCS4+ backbone with simian cytomegalovirus promoter and flanking multiple cloning sites. The 2A sequences included GSG linker additions at the N-terminus to improve cleavage efficiency. These vectors enabled stoichiometric co-expression of multiple proteins from a single open reading frame, overcoming IRES limitations of large size (>500 nucleotides) and unequal translation efficiency between upstream and downstream genes.

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Abstract

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.