Editor's Review,P2A is the optimal sequence for polycistronic expression

The 2A peptide cleavage mechanism has been a subject of intense research, particularly with significant advancements noted around 2017. These short, 18- to 22-amino-acid sequences, primarily derived from viruses like the Foot-and-Mouth Disease Virus (FMDV), are fascinating for their ability to facilitate the production of multiple distinct proteins from a single messenger RNA (mRNA) transcript. This process, often referred to as self-cleaving, is not a true enzymatic cleavage in the traditional sense but rather a remarkable ribosomal event.

At its core, the 2A peptide functions by interacting with the ribosome during translation. Specifically, it engages with the ribosome exit tunnel to inhibit the formation of a normal peptide bond at its C-terminus. This inhibition leads to a phenomenon known as ribosomal stalling and peptide bond skipping recoding protein translation. Instead of continuing to extend the polypeptide chain, the ribosome effectively skips the formation of the peptide bond between the glycine (Gly) residue and the subsequent proline (Pro) at the C-terminal end of the 2A peptide sequence. This "skip" results in the release of a completed upstream protein and the continuation of translation for the downstream protein(s).

The 2A peptide sequences allow a eukaryotic cell to produce multiple separated peptides from one mRNA through an event often described as "stop-and-carry-on." This ribosome skipping the formation of a glycyl-prolyl peptide bond is crucial for creating polycistronic expression systems, which are invaluable tools in molecular biology and genetic engineering. The 2A peptide acts as a molecular linker, mediating this co-translational cleavage of polyproteins.

While the general principle is understood, the exact molecular mechanism of 2A-peptide-mediated cleavage is still unknown in its entirety. However, research has elucidated key aspects. The peptide is "cleaved" C-terminal to the Gly residue in the PGP (Pro-Gly-Pro) sequence, a common motif within many 2A peptides. This results in the upstream protein terminating with a proline residue, while the downstream protein begins with the proline from the next codon. It's important to note that this process doesn't result in a complete separation; the 2A sequences will leave a peptide on the C-terminus of the upstream protein (typically a proline) and a single amino acid (often a proline) on the N-terminus of the downstream protein.

Several types of 2A peptides have been identified and characterized, each with varying efficiencies. Among these, the P2A sequence, derived from the Porcine teschovirus-1, has emerged as particularly effective. Studies, including those from 2017, have systematically compared different 2A peptides for their utility in cloning multi-gene constructs. Research has indicated that the P2A is the optimal sequence for polycistronic expression due to its high cleavage efficiency. For instance, the 19-amino acid P2A elicits higher cleavage efficiency than the 22-amino acid 2As, suggesting that sequence length and composition play significant roles.

The cleavage efficiency of 2A peptides depends on its amino acid sequence. Despite extensive study, there can be contrasting results present in the literature regarding the efficiency of specific 2A peptides. Factors such as the surrounding amino acid sequences of the fused proteins and the cellular context can influence the efficiency of the 2A peptide-mediated co-expression system. Researchers continue to explore optimizations to improve cleavage efficiency for these systems.

The concept of self-cleaving 2A peptide is widely used, but it's more accurately described as a ribosomal stalling and peptide bond skipping recoding protein event. It's not a process mediated by secreted enzymes or proteases. This distinction is critical for understanding its application and limitations.

The 2A peptide cleavage mechanism has opened avenues for creating synthetic polycistronic sequences in eukaryotes, enabling the simultaneous expression of multiple genes of interest. This is particularly useful for expressing functional proteins, such as those in antibody or enzyme production. The cleaving peptide acts as a ribosomal bypass signal, allowing for the ordered expression of genes within a single transcript.

In summary, the 2A peptide cleavage mechanism is a sophisticated ribosomal event that allows for the co-translational separation of proteins from a single mRNA. While the fundamental process involves ribosome skipping, the precise molecular details are still being uncovered. The ongoing research, with significant contributions noted around 2017, continues to refine our understanding and application of these powerful peptides in various biological and biotechnological fields. The 2A system, with its inherent self-cleaving 2A peptide activity, remains a cornerstone for efficient polycistronic gene expression.