Английская Википедия:2A self-cleaving peptides
2A self-cleaving peptides, or 2A peptides, is a class of 18–22 aa-long peptides, which can induce ribosomal skipping during translation of a protein in a cell.[1][2] These peptides share a core sequence motif of Шаблон:Tt, and are found in a wide range of viral families. 2A peptides are used to help generate polyproteins by causing the ribosome to fail at making a peptide bond.[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]
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.[1]
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 | Шаблон:Mono |
| P2A | Шаблон:Mono |
| E2A | Шаблон:Mono |
| F2A | Шаблон:Mono |
Description
The apparent cleavage is triggered by ribosomal skipping of the peptide bond between the proline (P) and glycine (G) in C-terminal of 2A peptide, resulting in the peptide located upstream of the 2A peptide to have extra amino acids on its C-terminal end while the peptide located downstream the 2A peptide will have an extra proline on its N-terminal end. 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 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, rescuing the function of the two peptides.[6]
2A peptides, when combined (or not) with the IRES elements, can make it possible to generate multiple separated peptides within a single transcript.[1]
Incomplete cleavage
Different 2A peptides have different efficiencies of self-cleaving, T2A and P2A being the most and F2A the least efficient.[11][12] 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.[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%.[14] 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.
See also
References
- ↑ 1,0 1,1 1,2 1,3 Шаблон:Cite journal
- ↑ Шаблон:Cite book
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ 6,0 6,1 Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite web
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
- ↑ Шаблон:Cite journal
