2APeptide

2A peptides, also known as 2A sequences, are short viral-derived peptide motifs used in molecular biology to produce multiple proteins from a single messenger RNA in eukaryotic cells. They enable polycistronic expression without requiring proteolytic enzymes, facilitating coordinated expression of several proteins from one transcription unit.

The mechanism is a ribosome skip during translation. Within the 2A sequence, the ribosome fails to form a peptide bond between two residues, typically a glycine and a proline, resulting in the upstream protein ending with a short 2A-derived tail and the downstream protein starting with a proline. This produces two discrete polypeptides from a single open reading frame, with the 2A sequence largely retained on the upstream protein.

Several natural 2A peptides are commonly used in research and biotechnology, including P2A (porcine teschovirus-1), T2A (Thosea asigna virus), E2A (equine rhinitis A virus), and F2A (foot-and-mouth disease virus). They vary in length and cleavage efficiency depending on the organism, cell type, and surrounding sequence. In practice, the choice of 2A peptide can influence the expression balance and may require testing to optimize performance for a given application.

Applications include co-expression of reporters and functional proteins, multi-subunit enzymes, or therapeutic constructs within a single vector. Compared with internal ribosome entry sites (IRES), 2A peptides often yield more balanced expression and simpler construct design, though there can be residual 2A-derived residues on the upstream protein, and downstream expression efficiency can be context-dependent. Careful design, including consideration of tags, localization signals, and codon optimization, is recommended.