Coding

Part:BBa_K3504018

Designed by: Ahmed Wael   Group: iGEM20_AFCM-Egypt   (2020-10-15)
Revision as of 09:04, 22 October 2020 by AhmedAdel01 (Talk | contribs) (→‎Usage)


P2A

Part Description

2A self-cleaving peptides is a class of 18–22 aa long peptides that can induce ribosomal skipping during translation of a protein in a cell.

Usage

2A peptides are capable of cleaving a large peptide into two shorter fragments. 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.

Characterization

P2A have been shown to have superior cleavage ability when compared to different 2A peptide.

Figure 1. Comparison of different 2A peptides according to trastuzumab expression.
















Improvements

We improved the part BBa K1537016 by Inserting GSG and SGS linkers, V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag immediately preceding 2A has been shown to improve 2A cleavage efficiency

Figure 1. P2A+GSG enhancement using V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag.







References

1.Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol 2004; 22:589-94; PMID:15064769; http://dx.doi.org/10.1038/nbt957

2.Yang S, Cohen CJ, Peng PD, Zhao Y, Cassard L, Yu Z, Zheng Z, Jones S, Restifo NP, Rosenberg SA, et al. . Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition. Gene Ther 2008; 15:1411-23; PMID:18496571; http://dx.doi.org/10.1038/gt.2008.90

3.Tan YP, Liang HR, Chen AE, Guo XF. Coexpression of double or triple copies of the rabies virus glycoprotein gene using a 'self-cleaving' 2A peptide-based replication-defective human adenovirus serotype 5 vector. Biologicals 2010; 38:586-93; PMID:20682459; http://dx.doi.org/10.1016/j.biologicals.2010.06.001

4.Chng, J., Wang, T., Nian, R., Lau, A., Hoi, K. M., Ho, S. C., ... & Yang, Y. (2015, March). Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. In MAbs (Vol. 7, No. 2, pp. 403-412). Taylor & Francis.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None