2A self-cleaving peptides


2A self-cleaving peptides, or 2A peptides, is a class of 18–22 aa-long peptides, which can induce the cleaving of the recombinant protein in a cell. These peptides share a core sequence motif of, and are found in a wide range of viral families. They help break apart polyproteins by causing the ribosome to fail at making a peptide bond.
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.

Members

Four members of 2A peptides family are frequently used in life science research. They are P2A, E2A, F2A and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; T2A is derived from thosea asigna virus 2A.
The following table shows the sequences of four members of 2A peptides. Adding the optional linker “GSG” on the N-terminal of a 2A peptide helps with efficiency.
NameSequence
T2A
P2A
E2A
F2A

Description

The cleavage is trigged by breaking of peptide bond between the Proline and Glycine in C-terminal of 2A peptide. The exact molecular mechanism of 2A-peptide-mediated cleavage is still unknown. However, it is believed to involve ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage.

Application

In genetic engineering, the 2A peptides are used to cleave a longer peptide into two shorter peptides. The 2A peptides can be applied when the fused protein doesn’t work. Inserting the CDS of a 2A peptide into the fusing point or replacing the linker sequence with the CDS of a 2A peptide protein can cleave the fused protein into two separated peptides, making the two peptides to regain the function.
2A peptides, when combined with the IRES elements, can make it possible to generate four separated peptides within a single transcript.

Incomplete cleavage

Different 2A peptides have different efficiencies of self-cleaving, P2A being the most and F2A the least efficient. Therefore, up to 50% of F2A-linked proteins can remain in the cell as a fusion protein, which might cause some unpredictable outcomes, including a gain of function.