We have fused a protein of interest with EGFP into plasmid vector under CMV promoter control. In WB we have got two bands correspond to EGFP itself and fused recombinant protein according to Mw. What is the reason for this independent expression or cleavage.
Assuming that it was against the tag on the FP side,
it is indeed some partial cleavage. (To distinguish
from alternative translation start, see if this depends
on incubation time)
Unfolded sequence between two proteins may be more vulnerable to that.
You could try to change the linker sequence between the
two proteins.
It is indeed Ab against full length GFP ( sc-8334).
BTW, there is no linker sequence in between.
That's reasonable Arjan. Thanks. Is there a way to check it alternatively? If there is a tiny contamination we will not be able to see it in restriction analysis?
- do you know the structure of the protein?
If there is no linker fusion may interfere with the folding
of either EFP or your protein.
Unfortunately structure is not known. We basically operate with predicted structure. Anyway, misfolding does not lead to expression of two proteins. I might wrong...
Misfolding can lead to cleavage for sure. Every GFP fusion construct should at least have a five amino acid linker. GFP structure and folding is very peculiar and might interfere with the folding of the other moiety (e.g. your protein of interest). You should also check wether fusing your protein in the other orientation could solve your problem. Some protein have lipid modification in N-ter or C-ter which could interfere with the proper folding and also lead to degradation product.
I would just take some EFP fusion construct that has worked
for similar purposes (I guess microscopy) and adapt it for your
project. This could save a lot of uncertainties.
Joanna, EGFP fused with C-term of our gene. We have some additional bands on WB. Some of them are lower Mw ( below 25 kDa) some are higher (50 kDa) than we expected to see.
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