EGFP cloning and membrane protein - (Dec/15/2008 )
Hi!
I want to clone a gene coding a membrane-bound protein in a EGFP vector (preferently, a EGFP-C vector, that is, my protein cloned in the N-terminal extrem of GFP protein). I am not sure it is wise to clone in this position a protein which would be bound in the membrane. Could it affect the correct localization of my protein? Do you think it would be better to try to clone it in the C-terminal position?
Any experience with GFP cloning of membrane-bound proteins?
Thanks!
I want to clone a gene coding a membrane-bound protein in a EGFP vector (preferently, a EGFP-C vector, that is, my protein cloned in the N-terminal extrem of GFP protein). I am not sure it is wise to clone in this position a protein which would be bound in the membrane. Could it affect the correct localization of my protein? Do you think it would be better to try to clone it in the C-terminal position?
Any experience with GFP cloning of membrane-bound proteins?
Thanks!
Hi
You should analyze the structure of your protein first, in order to find whether there are some specific domains within the protein sequences,just as tran-membrane, signal-peptite and so on. Then you can determine the fusion position and prevent to destroy the specific structure.
Stone
Hi E.G.
I have worked a lot with polarized membrane proteins tagged with YFP, CFP, GFP (... and so on..) and for experience the best way to know it is producing both constructs and then try it in your cells; some cell lines are far more stringent in terms of quality control during protein folding and membrane delivery than others. What you should always care is that the membrane targeting sequence in your protein is not modified. In addition, sometimes it requires and scaffolding partner (or even an extracellular ligand) to stay in the membrane.
A couple of questions that could help:
What is the purpose of tagging the protein ? in some cases, like FRET or FLIM, the position of the fluorophore is critical.
Do the protein has interaction partners ? (if yes.... is the C-terminus or N-terminus involved ?)
Is the C-terminal or N-terminal sequence highly conserved among species ? (if so, it is more likely that modifying the region in question would lead to an abnormal expression pattern)
Is the cell line I'm using stringent in terms of membrane delivery and polarity ? (some epithelial and endothelial cells simply will not deliver the constructs to the membrane)..... if so consider using a more flexible cell line like HEK's
