We are trying to use the P2A self-cleavage peptide to produce equimolar expression of 2 protein. However, we can't get the protein cleaved, we only detect the uncleaved product. The protein sequence is correct: GSGATNFSLLKQAGDVEENPGP .
While looking at the literature, I realized that the mechanism is not well understood. Some propose protease cleavage and other ribosome skip...
My question is simple: Is the DNA sequence is important or not?
In my case, we just got the protein sequence codon optimized and synthesized.
Thanks for your input!
Patrick M Giguère did you manage to solve the problem? It seems we experience the same problem for some protein pairs. Is the DNA sequence important? In one paper I did read the DNA sequence of the conserved motif is important not to codon optimize, but on the other hand I find a lot of plasmids on addgene with P2A linkers that are codon optimized, also in the motif region.
Hi, I have been struggling with this same problem in the past month or so and I just found this paper:
75593028 (cshlp.org)
Which it seems to suggest that the original codon choice is important for the efficiency in ribosome skipping. Paradoxically, this could be one of the few counterintuitive occasions where codon optimization is making your life harder rather than easier.
I just noticed that the link is not sending you in the right place. The paper is Kjaer and Belsham 2018 (RNA). Below the link on Pubmed
Article Selection of functional 2A sequences within foot-and-mouth d...
- Badges
- Science method