2A peptides explained

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, 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.

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
P2A
E2A
F2A

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.

See also

Notes and References

  1. Liu Z, Chen O, Wall JB, Zheng M, Zhou Y, Wang L, Vaseghi HR, Qian L, Liu J . 6 . Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector . Scientific Reports . 7 . 1 . 2193 . May 2017 . 28526819 . 5438344 . 10.1038/s41598-017-02460-2 . 2017NatSR...7.2193L .
  2. Book: Sampath Karuna. Roy Sudipto. Live Imaging In Zebrafish: Insights Into Development And Disease. 30 August 2010. World Scientific. 978-981-4464-89-5. 51–52.
  3. Luke GA, de Felipe P, Lukashev A, Kallioinen SE, Bruno EA, Ryan MD . Occurrence, function and evolutionary origins of '2A-like' sequences in virus genomes . The Journal of General Virology . 89 . Pt 4 . 1036–1042 . April 2008 . 18343847 . 2885027 . 10.1099/vir.0.83428-0 .
  4. Yang X, Cheng A, Wang M, Jia R, Sun K, Pan K, Yang Q, Wu Y, Zhu D, Chen S, Liu M, Zhao XX, Chen X . 6 . Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins . Frontiers in Microbiology . 8 . 1373 . 2017 . 28785248 . 5519566 . 10.3389/fmicb.2017.01373 . free .
  5. Ryan MD, King AM, Thomas GP . 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 . November 1991 . 1658199 . 10.1099/0022-1317-72-11-2727 . free .
  6. 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 . 10.1101/pdb.ip067876 . 22301656.
  7. Wang Y, Wang F, Wang R, Zhao P, Xia Q . 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori . Scientific Reports . 5 . 1 . 16273 . November 2015 . 26537835 . 4633692 . 10.1038/srep16273 . 2015NatSR...516273W .
  8. Web site: Cleavage Activity of Aphtho- and Cardiovirus 2A Oligopeptidic Sequences.. University of St Andrews. 2019-01-05. https://web.archive.org/web/20161230094902/http://www.st-andrews.ac.uk/ryanlab/page2.htm. 2016-12-30. dead.
  9. Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD . 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 . May 2001 . 11297676 . 10.1099/0022-1317-82-5-1013 . free .
  10. Sharma P, Yan F, Doronina VA, Escuin-Ordinas H, Ryan MD, Brown JD . 2A peptides provide distinct solutions to driving stop-carry on translational recoding . Nucleic Acids Research . 40 . 7 . 3143–51 . April 2012 . 22140113 . 3326317 . 10.1093/nar/gkr1176 .
  11. Chng J, Wang T, Nian R, Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y . 6 . Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells . mAbs . 7 . 2 . 403–12 . 2015-03-04 . 25621616 . 4622431 . 10.1080/19420862.2015.1008351 .
  12. Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, Kim MK, Shin BA, Choi SY . 6 . High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice . PLOS ONE . 6 . 4 . e18556 . 2011-04-29 . 21602908 . 3084703 . 10.1371/journal.pone.0018556 . Volker . 2011PLoSO...618556K . Thiel . free .
  13. Velychko S, Kang K, Kim SM, Kwak TH, Kim KP, Park C, Hong K, Chung C, Hyun JK, MacCarthy CM, Wu G, Schöler HR, Han DW . 6 . Fusion of Reprogramming Factors Alters the Trajectory of Somatic Lineage Conversion . Cell Reports . 27 . 1 . 30–39.e4 . April 2019 . 30943410 . 10.1016/j.celrep.2019.03.023 . free .

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