The P2A sequence is a short peptide used in molecular biology to manage the expression of multiple proteins from a single genetic message. This sequence allows researchers to ensure that several proteins are created simultaneously and in approximately equal amounts within a cell. This method of co-expression offers an advantage over other techniques due to the P2A sequence’s small size and high efficiency in separating the resulting proteins. The utility of this sequence centers on its unique ability to manipulate the cell’s natural protein-making machinery, making it useful for gene therapy vectors and genetic studies.
The Sequence Structure and Origin
The P2A sequence belongs to the family of 2A peptides, which originated from the genomes of positive-sense single-stranded RNA viruses. P2A specifically derives from the Porcine Teschovirus-1, which is the source of its designation. The sequence is short, typically composed of 18 to 22 amino acids, and is translated as part of a single, continuous polypeptide chain.
The functional core of the P2A sequence is a conserved C-terminal motif, including the amino acid pattern D-E-N-P-G-P. The final two residues, a Glycine (G) followed by a Proline (P), are structurally necessary for the peptide’s unique mechanism. The 2A family also includes:
- F2A, sourced from Foot-and-Mouth Disease Virus.
- T2A, sourced from Thosea asigna virus.
- E2A, sourced from Equine Rhinitis A virus.
Researchers sometimes add a small Glycine-Serine-Glycine (GSG) linker immediately before the P2A sequence to enhance its functional efficiency.
The Unique Ribosomal Skipping Mechanism
The mechanism by which the P2A sequence separates proteins is known as ribosomal skipping or “stop-carry on” translation, which is not true enzymatic cleavage. When the ribosome translates the P2A coding sequence, it encounters a specific point between the conserved Glycine and Proline residues. At this junction, the ribosome fails to form the peptide bond that would normally link the Glycine residue to the following Proline residue.
Instead, the peptidyl-tRNA ester linkage connecting the nascent polypeptide chain to the tRNA in the P-site is hydrolyzed. The peptidyl-transferase center of the ribosome is thought to undergo a structural modification induced by the P2A sequence. The upstream protein is released from the ribosome, and the translation machinery then reinitiates the synthesis of the downstream protein.
The result of this skipping event is two separate protein products from the single mRNA transcript. The upstream protein retains the entire P2A sequence, ending with the conserved C-terminal Glycine residue. The downstream protein begins with the Proline residue that immediately followed the Glycine in the P2A sequence. This co-translational separation occurs without the need for an additional protease enzyme.
Essential Applications in Genetic Engineering
The P2A sequence enables the creation of polycistronic vectors in eukaryotic cells, which typically only translate one protein per messenger RNA molecule. By inserting P2A between the coding regions of multiple genes, researchers can express several proteins from a single promoter and mRNA transcript. This ensures all genes are expressed simultaneously and at a roughly equal level.
A significant application is in gene therapy, particularly with viral vectors like adeno-associated viruses (AAV), where the capacity for genetic material is severely limited. The P2A sequence is much shorter than other co-expression elements, such as the Internal Ribosomal Entry Site (IRES), allowing more therapeutic genes to be packaged into the vector.
The sequence is also used to build reporter systems, where the expression of a difficult-to-detect protein is directly linked to an easily visualized marker, like Green Fluorescent Protein (GFP). This linked expression provides a simple way to track cell transfection and confirm that the therapeutic gene is being made.
Assessing Cleavage Efficiency and Fidelity
Ribosomal skipping is rarely 100% efficient in practice, leading to a phenomenon known as “read-through” or “fusion product.” In read-through events, a small percentage of ribosomes fail to skip the peptide bond formation and instead produce a single, long fusion protein. This uncleaved product contains the upstream protein, the full P2A sequence, and the downstream protein.
This fusion product can be non-functional, and in gene therapy contexts, the foreign viral peptide sequence can potentially trigger an unwanted immune response in the host. P2A efficiency is high compared to other 2A peptides, showing superior performance across different cell types, including human cell lines, zebrafish, and mice.
Cleavage efficiency is calculated by comparing the amount of successfully separated protein products to the total amount of protein produced, including the fusion product. Researchers assess this fidelity using laboratory techniques such as Western blotting, which separates and quantifies the cleaved and uncleaved protein forms based on their distinct molecular weights. Optimizing the P2A system may involve testing the sequence in tandem with other 2A peptides.