2A peptides
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.
2A function in FMVD virus
Foot-and-mouth disease virus (FMDV) is a virus in the genus Aphthovirus that causes foot-and-mouth disease in cattle.[6] As a member of the family Picornaviridae, FMDV is a positive-sense, single-stranded RNA virus.
2A is located between the capsid proteins and polymerases and proteases required for the capsid assembly in the late stages of the infection. The incomplete ribosomal skipping during the viral RNA translation mediated by the 2A results in "ribosome drop off" in some cases, after which the translation doesn't restart. This leads to a prevalence of the N-terminal proteins and allows for the increased production of the structural proteins relative to the non-structural proteins.[7]
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.[8]
| Name | Sequence |
|---|---|
| T2A | (GSG) EGRGSLLTCGDVEENPGP |
| 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.[9][10] However, it is believed to involve ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage.[11][12]
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.[13][14] 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.[15] 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.
See also
References
- ^ a b c d Liu Z, Chen O, Wall JB, Zheng M, Zhou Y, Wang L, et al. (May 2017). "Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector". Scientific Reports. 7 (1) 2193. Bibcode:2017NatSR...7.2193L. doi:10.1038/s41598-017-02460-2. PMC 5438344. PMID 28526819.
- ^ Sampath Karuna; Roy Sudipto (30 August 2010). Live Imaging In Zebrafish: Insights Into Development And Disease. World Scientific. pp. 51–52. ISBN 978-981-4464-89-5.
- ^ Luke GA, de Felipe P, Lukashev A, Kallioinen SE, Bruno EA, Ryan MD (April 2008). "Occurrence, function and evolutionary origins of '2A-like' sequences in virus genomes". The Journal of General Virology. 89 (Pt 4): 1036–1042. doi:10.1099/vir.0.83428-0. PMC 2885027. PMID 18343847.
- ^ Yang X, Cheng A, Wang M, Jia R, Sun K, Pan K, et al. (2017). "Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins". Frontiers in Microbiology. 8 1373. doi:10.3389/fmicb.2017.01373. PMC 5519566. PMID 28785248.
- ^ Ryan MD, King AM, Thomas GP (November 1991). "Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence". The Journal of General Virology. 72 ( Pt 11) (11): 2727–32. doi:10.1099/0022-1317-72-11-2727. PMID 1658199.
- ^ Carrillo C, Tulman ER, Delhon G, et al. (May 2005). "Comparative Genomics of Foot-and-Mouth Disease Virus". J. Virol. 79 (10): 6487–504. doi:10.1128/JVI.79.10.6487-6504.2005. PMC 1091679. PMID 15858032.
- ^ Donnelly, M. L. L., Luke, G. A., Mehrotra, A., Li, X., Hughes, L. E., Gani, D., & Ryan, M. D. (2001). "Analysis of the aphthovirus 2A/2B polyprotein "cleavage" mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal "skip."". Journal of General Virology. 82 (5): 1013–1025. doi:10.1099/0022-1317-82-5-1013. PMID 11297676. Retrieved 3 September 2025.
{{cite journal}}: CS1 maint: multiple names: authors list (link) - ^ 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. PMID 22301656.
- ^ Wang Y, Wang F, Wang R, Zhao P, Xia Q (November 2015). "2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori". Scientific Reports. 5 (1) 16273. Bibcode:2015NatSR...516273W. doi:10.1038/srep16273. PMC 4633692. PMID 26537835.
- ^ "Cleavage Activity of Aphtho- and Cardiovirus 2A Oligopeptidic Sequences". University of St Andrews. Archived from the original on 2016-12-30. Retrieved 2019-01-05.
- ^ Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD (May 2001). "Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'". The Journal of General Virology. 82 (Pt 5): 1013–1025. doi:10.1099/0022-1317-82-5-1013. PMID 11297676.
- ^ Sharma P, Yan F, Doronina VA, Escuin-Ordinas H, Ryan MD, Brown JD (April 2012). "2A peptides provide distinct solutions to driving stop-carry on translational recoding". Nucleic Acids Research. 40 (7): 3143–51. doi:10.1093/nar/gkr1176. PMC 3326317. PMID 22140113.
- ^ Chng J, Wang T, Nian R, Lau A, Hoi KM, Ho SC, et al. (2015-03-04). "Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells". mAbs. 7 (2): 403–12. doi:10.1080/19420862.2015.1008351. PMC 4622431. PMID 25621616.
- ^ Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. (2011-04-29). Thiel V (ed.). "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice". PLOS ONE. 6 (4) e18556. Bibcode:2011PLoSO...618556K. doi:10.1371/journal.pone.0018556. PMC 3084703. PMID 21602908.
- ^ Velychko S, Kang K, Kim SM, Kwak TH, Kim KP, Park C, et al. (April 2019). "Fusion of Reprogramming Factors Alters the Trajectory of Somatic Lineage Conversion". Cell Reports. 27 (1): 30–39.e4. doi:10.1016/j.celrep.2019.03.023. PMID 30943410.