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