High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice

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.

Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit

“…Consistent with previous studies, we observed a high amount of protein degradation of the P2A* product , which correlated with the decrease in expression observed using confocal microscopy ( Figure S2). A barely detectable band that could correspond to a potential uncleaved product was sometimes observed using the a-mCherry antibody in T2A, E2A, and P2A lines ( Figure 1C), suggesting that, for these 2A viral peptides, a small residual fraction of noncleaved product may exist, as reported in some other models (Kim et al 2011). In sum, we found that each of the canonical F2A, T2A, E2A, and P2A viral peptides work efficiently in C. elegans.…”

“…Because an uncleaved product can sometimes be detected, but not always, as already described in other species (Hasegawa et al 2007;Provost et al 2007;Gonzalez et al 2011;Kim et al 2011), we cannot exclude that, in rare cases, low amounts of an uncleaved product could be associated with a dominant negative activity. It should be noted that this has never been described, while the multi-subunit T-cell receptor, or cocktails of pluripotency factors, for example, have been delivered to cells with no adverse effects (Szymczak et al 2004;Carey et al 2009).…”

“…It should be noted that this has never been described, while the multi-subunit T-cell receptor, or cocktails of pluripotency factors, for example, have been delivered to cells with no adverse effects (Szymczak et al 2004;Carey et al 2009). Different efficiencies have been reported for a given 2A peptide in different species (Kim et al 2011). This could be due to the phylogenetic distance between the species naturally infected by the 2A-containing virus and the species tested.…”

Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System

“…To determine the feasibility of our KI approach, the GFP coding region was fused in frame with the selection marker Neomycine phosphotransferase II (NPTII) using the self-cleaving peptide 2A (Kim et al, 2011) as a bridge (Supplemental Figure 4). The translation start codon "ATG" was removed from this fusion cassette in order to inhibit any undesired translation caused by random insertion of the T-DNA into the rice genome.…”

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

“…The use of 2A variants can avoid potential intramolecular homologous recombination of polycistrons, and allows for the expression of multiple genes (up to five genes). There are four widely used 2A sequences, which are derived from the foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), Thoseaasigna virus (T2A), and equine rhinitis A virus (E2A), with P2A as the most effective one [98]. 2A is active in all eukaryotic cells [99], but not in prokaryotes [100].…”