Difference between revisions of "Part:BBa K4140004"

Latest revision as of 17:04, 10 October 2022

P2A linker

Part Description

In order to mediate the "cleavage" of two proteins, 2A peptide sequences were discovered in Picornaviruses. Effective multiple protein expression from a single open reading frame (ORF) is achieved using 2A peptide-linked multicistronic vectors. Use alternative 2A peptide sequences when linking more than two genes to reduce the likelihood of homologous recombination. All eukaryotic systems studied to date, including mammalian cells, yeast, and plants, have effectively utilised the 2A peptide system.

Usage

Before implementing our therapeutic circuit product into a human body, we employed this 2A linker to attach a reporter gene to it so that we could test its functionality and ability to express PAH by utilizing spectrophotometry to monitor the degree of PAH expression.

Literature Characterization

In this study, they found that among four different 2A peptides, a 2A peptide derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in three human cell lines (HEK293T,HT1080,HeLa)

Figure 1. P2A shows the highest cleavage efficiency in HEK293T cells.

Figure 2. P2A shows the highest cleavage efficiency in HeLa cells

Figure 4. Construction of expression plasmids harboring DNA sequences encoding various 2A peptides flanked by multiple cloning sites.

Figure 3. P2A shows the highest cleavage efficiency in HT1080 cells

Comparison between P2A and Internal ribosome entry site (IRES) as Bicistronic or multicistronic expression vectors: Bicistronic or multicistronic expression vectors have been used to express more than one gene in a cell. A new approach was needed to replace IRES because of the magnitude and variation in expression levels between the genes before and after IRES. That’s why P2A peptide, a self-cleaving 2A peptide would be a potential replacement for IRES due to its small size and great effectiveness of cleavage between genes upstream and downstream of the P2A.

Experimental Characterization

This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 5. The running part (ordered from IDT) included ParoF promoter - P2A - L7Ae.

References

1. Kim, J. H., Lee, S. R., Li, L. H., Park, H. J., Park, J. H., Lee, K. Y., ... & Choi, S. Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PloS one, 6(4), e18556.‏

Sequence and Features