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==Application==
==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 [[Coding DNA sequence|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.<ref name="Szymczak-WorkmanVignali2012">{{cite journal | vauthors = Szymczak-Workman AL, Vignali KM, Vignali DA | title = Design and construction of 2A peptide-linked multicistronic vectors | journal = Cold Spring Harbor Protocols | volume = 2012 | issue = 2 | pages = 199–204 | date = February 2012 | pmid = 22301656 | doi = 10.1101/pdb.ip067876 }}</ref>
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 [[Coding DNA sequence|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.<ref name="Szymczak-WorkmanVignali2012">{{cite journal | vauthors = Szymczak-Workman AL, Vignali KM, Vignali DA | title = Design and construction of 2A peptide-linked multicistronic vectors | journal = Cold Spring Harbor Protocols | volume = 2012 | issue = 2 | pages = 199–204 | date = February 2012 | pmid = 22301656 | doi = 10.1101/pdb.ip067876 }}</ref>


2A peptides, when combined (or not) with the [[Internal ribosome entry site|IRES]] elements, can make it possible to generate multiple separated peptides within a single [[Primary transcript|transcript]].<ref name="LiuChen2017"/>
2A peptides, when combined (or not) with the [[Internal ribosome entry site|IRES]] elements, can make it possible to generate multiple separated peptides within a single [[Primary transcript|transcript]].<ref name="LiuChen2017"/>

Revision as of 23:05, 21 June 2022

An illustration of 2A peptide function: when the CDS of a 2A peptide is inserted between two CDSs of protein, the peptide will be translated into two proteins folding independently due to ribosome skipping (not due to cleavage as shown in the figure).

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 DxExNPGP, and are found in a wide range of viral families. They help generating 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 (GSG) EGRGSLL TCGDVEENPGP
P2A (GSG) ATNFSLLKQAGDVEENPGP
E2A (GSG) QCTNYALLKLAGDVESNPGP
F2A (GSG) VKQTLNFDLLKLAGDVESNPGP

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. ^ 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.
  2. ^ 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.
  3. ^ 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.
  4. ^ 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.
  5. ^ 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.
  6. ^ a b 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.
  7. ^ 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.
  8. ^ "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.
  9. ^ 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.
  10. ^ 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.
  11. ^ 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.
  12. ^ 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.
  13. ^ 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.
  14. ^ 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.
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