Hi there. I'm expressing a construct (CMV-proteinA-P2A-ProteinB) in human cell line system. I have 3 questions.
1. I know 2A peptide is called "self-cleavage peptide". But is there any involvement of protease? And what's the mechanism of the cleavage.
2. Does anyone know exactly where on those 10-ish amino acids of P2A get cleaved? Is there going to be any residues on the C-terminal of my protein A and N-terminal of protein B?
3. How's the cleavage efficiency? Do they got cleaved right after translation? Do they all get cleaved or there is a small fraction don't?
Thank you all
Zach
The peptide is "cleaved" C-terminal to the Gly residue in the PGP sequence, leaving an N-terminal proline attached to the C-terminal polypeptide and the rest of the 2A peptide attached to the C-terminus of the N-terminal polypeptide.
The mechanism of cleavage is thought not to be proteolytic, but to be caused by "ribosome skipping," which means that the ribosome moves from one codon to the next without forming a peptide bond. In other words, the "cleavage" occurs during translation, but it isn't really cleavage.
More information can be read here:
http://blog.allelebiotech.com/tag/2a-peptide/
and here
https://www.nature.com/articles/s41598-017-02460-2
and here
Article Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mec...
Adam B Shapiro is correct there is no actually cleavage they are more appropriately called "ribsomal skipping peptides". Just to add to the above answer:
1- there will be residues on both proteins (the kind of residue depends on which peptide you used)
2- if you look at the papers using such peptides. They usually calculate the "cleavage efficiency" in a given system by comparing multiple peptides like T2A and such. Depending on your system different peptides give different efficiencies like 85% cleavage and it must be assessed in each given system. The skipping occur during translation and some of the transcripts will fail to skip and you will end up with a chimeric protein.
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