N terminal GFP fusions - is it as appropriate as C terminus? (Apr/28/2008 )
Hey all.
I am interested to investigate localistion of my fav protein. Due to previous experimental design and financial and time constraints, I have the best option of an N terminal GFP tag as opposed to C terminal, driven by the native promoter. Does anyone have experience with this? I notice the "standard" is a C terminal tag.... is a 6ish nucleotide linker enuf to ensure correct folding of my protein or do i need a longer linker?
thanks heaps in advance for your advice with this.
fatty
I am interested to investigate localistion of my fav protein. Due to previous experimental design and financial and time constraints, I have the best option of an N terminal GFP tag as opposed to C terminal, driven by the native promoter. Does anyone have experience with this? I notice the "standard" is a C terminal tag.... is a 6ish nucleotide linker enuf to ensure correct folding of my protein or do i need a longer linker?
thanks heaps in advance for your advice with this.
fatty
I've had success, and issues, with both N terminal and C terminal so I think that it really depends on the structure of your protein. The more that you know about it, the better you can design the right placement of the GFP. If you can do the N terminal with little effort, I would say make that first and in the meantime have a backup plan to make the C terminal. It won't hurt to have both and you might find that one expresses better than the other.
The beauty of a C-terminal GFp is that you can see if the tag is correctly folded, and that is used as an assumption that the protein preceding it is also correctly folded.
cool thanks very much for your replies. I will start with the N term and in my "spare time" THINK about the C term!! fingers crossed it will all work out.....