Difference between revisions of "Part:BBa K1442039"

Revision as of 00:01, 21 October 2019

P2A self-cleaving peptide sequence

The self-cleaving 2A peptide (18-22 amino acids) is a virally derived coding region that has been utilised by viruses to self-cleave during translation and is likely to have arisen to overcome traditional IRES sequences due to its much smaller coding length and allows for a smaller viral genome [1].

Of the available 2A coding regions coding for the peptide from various viruses: foot-and-mouth disease virus, equine rhinitis A, Thosea asigna, porcine teschovirus-1. The porcine teschovirus 2A (P2A) had the highest efficiency of cleavage in three mammalian cell lines tested by Kim et al [1]: human, zebrafish and adult mice.

The P2A peptide can be used in a research context to allow multicistronic expression of genes without traditional methods of: multiple promoters, insertion of a splicing signal, insertion of a proteolytic cleavage site (e.g. TEV) and IRESs. Of note, P2A overcomes the limitations of an IRES: it is much smaller and also improves the translational efficiency of IRES-based genes [1]. The use of P2A is therefore useful to allow coexpression of large proteins in plasmids where the size of the insert is limiting and preserves the authenicity of a expressed protein sequence over traditionally cleaved protein in vitro such as TEV protease sites that leave a scar site.

Usage and Biology

The P2A sequence was added to our replicon between the MS2 coat protein and RdRp to separate it after translation from an upstream IRES, the P2A acts to reduce the size of the insert, reducing synthesis costs and complications in transformation and transfection.

Characterization by CPU_CHINA 2019

Figure 1.Flow cytometry analysis of TLR1 and TLR2 expression.

In order to determine the cleavage efficiency of P2A, 2019 CPU_CHINA iGEM team constructed TLR1 <a href="https://parts.igem.org/Part:BBa_K2976001" target="_blank">(BBa_K2976001)</a>, P2A self-cleaving peptide sequence <a href="https://parts.igem.org/Part:BBa_K1442039" target="_blank">(BBa_K1442039)</a> and TLR2 <a href="https://parts.igem.org/Part:BBa_K2976002" target="_blank">(BBa_K2976002)</a> together. Then, we integrated them into pcDNA6 vector and transfected the vector into HEK293T cells for co-expression of TLR1 and TLR2. The figure shows that the amount of TLR1 (A, B) and TLR2 (C, D) in the cells separately increased after transfection, which demonstrates that P2A exhibits efficient self-cleavage activity.

Sequence and Features