An illustration of 2A peptide function: when the CDS of a 2A peptide is inserted between two coding sequences, the peptide will be translated into two unbound proteins, due to ribosome skipping (i.e. not due to cleavage as shown in the figure).
2A peptides are a class of 18–22 aa-long peptides, which can induce ribosomal skipping during translation of a protein in a biological cell.[1][2] These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. 2A peptides can be introduced artificially to help generate polyproteins from a single ORF, by causing the ribosome to fail at making a peptide bond, and then resume translation.[3][4]
The members of 2A peptides are named after the virus in which they have been first described. For example, F2A, the first described 2A peptide, is derived from foot-and-mouth disease virus. The name "2A" itself comes from the gene numbering scheme of this virus.[1][5]
These peptides are also known as "self-cleaving" peptides, which is a known misnomer, because the missing peptide bond is never synthesized by the ribosome, and is thus not cleaved.
The following table shows the sequences of four members of 2A peptides. Adding the optional linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.[6]
Name
Sequence
T2A
(GSG) EGRGSLL TCGDVEENPGP
P2A
(GSG) ATNFSLLKQAGDVEENPGP
E2A
(GSG) QCTNYALLKLAGDVESNPGP
F2A
(GSG) VKQTLNFDLLKLAGDVESNPGP
Description
2A peptides trigger the ribosome to skip peptide bond formation between the glycine (G) and proline (P) near the C-terminus of the 2A peptide, resulting in the peptide located upstream of the 2A peptide having extra amino acids appended to its C-terminus while the protein downstream the 2A peptide will have an extra proline on its N-terminus. The exact molecular mechanism of 2A-peptide-mediated cleavage is still unknown.[7][8] However, it is believed to involve ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage.[9][10]
Application
In molecular biology, 2A peptides are used to express two separate proteins from a single open-reading frame. 2A peptides can be used when direct protein fusion does not work or is undesirable.
Efficiency of bond-skipping
Different 2A peptides have different peptide-bond-skipping efficiencies, with T2A and P2A being the most efficient and F2A the least efficient.[11][12] Therefore, up to 50% of F2A-linked proteins can in fact be produced as a fusion protein, which might cause some unpredictable outcomes, including a gain of function.[13] One study reported that 2A sites cause the ribosome to fall off approximately 60% of the time, and that, together with ribosome read-through of about 10% for P2A and T2A, this results in reducing expression of the downstream peptide chain by about 70%.[1] However, the level of drop-off detected in this study varied widely depending on the exact construct used, with some constructs showing little evidence of drop-off; furthermore, within a tri-cistronic transcript it reported a higher level of ribosome drop-off after one 2A sequence than after two 2As combined, which is at odds with a linear model of translation.
^Szymczak-Workman AL, Vignali KM, Vignali DA (February 2012). "Design and construction of 2A peptide-linked multicistronic vectors". Cold Spring Harbor Protocols. 2012 (2): 199–204. doi:10.1101/pdb.ip067876. PMID22301656.
