Expression of GFP fusion: splicing out of tag ? - (Jun/19/2006 )
I'm trying to express a construct composed of my protein of interest with a C-terminal GFP tag in tobacco. I got the construct and sequenced it and everything is OK (in frame, no stop codon,etc...). When I express this construct in N.benth using transient infection with agro, I can't detect any GFP (western or UV). I did a RT-PCR on RNA isolated from the infected tissu and I can detect the region coding for my protein but the portion coding for the GFP seems to be missing or really reduced in size (not sure if it's a PCR artifact). I don't know how to explain the disappearance of the GFP portion in the mRNA when it's present in the DNA. The only explanation I can think of is that I somehow introduce splicing sites during the cloning that removes the GFP. Another possibility is that some recombination event might have happen in Agro leading to removal of the GFP from the construct, but I've repeated the Agro transformation several independant times and they all give me the same result. I would really appreciate any advices that could help me deal with that issue.
Let me make myself clear first, this is translational fusion, so genomic DNA for that protein sequence with C terminal GFP sequence, right?
If this is the case, have you looked into the folding patterns of the protein that you are trying to express because sometimes it happens that because of vicinity of GFP, your protein won't fold properly and then for some reason GFP doesn't show up either.
I was suggested to put some extra Gs in between in-frame the genomic region of the DNA of interest and the coding region of GFP, also we have home-made vector which has some UTR region for GFP which stabilizes GFP mRNA. Check if you have such thing with the vector you are using. (If you have already used this vector successfully with any other protein then ignore this comment)