fluorescent tag on membrane protein - (Feb/27/2014 )
I'm trying to conjugate GFP on my target membrane protein in overexpression plasmid.
While searching for published plasmid data, i came across a plasmid which have inserted GFP after predicted signal peptide cleavage site as anything upstream of that will be cleaved off in ER.
so my question is...
1) is it really necessary to insert my GFP at that specific site of "after predicted signal peptide cleavage site" or isn't it entirely possible to simply insert the GFP at the 3' end of the protein?
2) how would yo go about finding this exact signal peptide cleavage site?
any comments appreciated!
Those plasmids usually use a 2A sequence which is cleaved at translation - so intact mRNA then cleaved into GFP and insert protein. You can conjugate the GFP directly to your protein if you like, but then you have to ensure that the GFP doesn't have the cleavage site between it and the insert protein. In which case it may well be easier to find a normal GFP cloning plasmid.
Check the sequence of the plasmid for the signal peptide sequence.
Important to know is the predicted topology of your membrane protein. For most membrane proteins, you will need to have your GFP at the c-terminus (3' end of your gene) because, as you are learning, signal peptides, signal sequences, etc at the Nterm are important for properly embedding the protein in the membrane. Disrupting that with a GFP gene will cause issues with expression.
Worst case scenario, buy a bunch of primers (primers are cheap) to put the GFP at the N-term (before/after signal sequence) and at C-term. Clone, express and put cells into fluorimeter. Which ever has fluorescence is your solution.
As an aside, beware that cloning is the easy part of working with membrane proteins. Once you get expression, then the real work begins. ;)