We are cloning a gene of interest (GOI) after P2A that is preceded by GFP. After P2A sequence there is a gap of few base pairs followed by gene of interest but this is in-frame with P2A and is translated as a single protein. My question is will there be any effect of those base pairs between P2A and GOI on the protein function?
As soon as you are in frame and the N-ter of your GOI is not totaly inside the molecule but in some extent exposed to solvent that should work
It depends, really. In many cases it will work, but suppose the P2A-GOI linker codes for a cysteine, or for a stretch of very hydrophobic side chains...
Also -and this has nothing to do with the linker- if the protein encoded by the GOI has to oligomerize or interact with other proteins to carry out its natural function, then having to carry around the additional mass represented by GFP and P2A will change the kon and therefore, binding affinity. Whether the change will be large enough to affect the biological function is another question, though.
You have to try and see.
Your resulting protein will contain few additional amino acids on N-terminal. This addition may sometimes affect the protein structure. If your protein has some signal sequence on N-term, this N-tail may cause problems with trafficking of protein in cell.
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