2A peptides are self-cleaving peptides 18-22 amino acids long that specify ribosome skipping during translation. These peptides were first identified in the oligopeptide sequence of foot-and-mouth disease virus (FMDV) RNA virus and are found in many other picornavirus families. They have been used to co-express multiple proteins in a single open reading frame by cleavage through a sequence of ribosomal skipping events within the polyprotein.
The principle, mechanism and practical use of a 2A peptide-based co-expression system is illustrated in Fig. 1. FMDV contains a large polyprotein, which can be divided into three regions: P1, P2, and P3 (Palmenberg 1987). Each of these regions is a contiguous coding sequence encoding one or more endogenous FMDV proteins with a unique 2A peptide positioned between each protein.
When a 2A peptide is placed at the 2A-to-B junction of the polyprotein, a ribosome skipping event occurs. This results in the formation of independent eukaryotic proteins from the polyprotein, which can be translated in parallel with the upstream FMDV protein. In addition to facilitating the expression of multiple endogenous proteins, the ribosome skipping event also allows for co-translational cleavage of non-structural proteins.
However, a number of limitations prevent the 2A-mediated co-expression system from being used widely. These include the lack of knowledge about the cleavage efficiency of different 2As in bi-cistronic constructs, as well as the effect of positional variations on gene expression. In addition, there is no widely-adopted cloning vector containing 2A-derived peptides for polycistronic expression.