My target protein is a secreted protein. I would like to track the secretion process of this protein. But the GFP fusion protein (GFP is in C terminal of my target protein) is not secrected out of cell by time lapse. Why is this the case?
If you fuse GFP to a signal peptide for translocation across the ER membrane, it will be very slowly secreted and you can see a lot of it in the ER waiting to be exported. This is because it interacts with the chaperone BiP.
If you have a soluble secreted protein, then you will also have an N-terminal signal peptide, and so it was a good start to place your GFP at the C-terminus, but there is no guarantee that the fusion protein is correctly folded. It is possible that the C-terminus of your protein of interest is normally located inside the folded protein or that it plays an important role in the overall structure for a different reason. In both cases, adding a large protein to the C-terminus like GFP may stop your protein from folding correctly. If that is the case, it should bind more strongly to BiP than just the signal peptide fusion to GFP (which is your control). You can test this by reciprocal immunoprecipitation:
1) use BiP antibodies (i.e. made in rabbit) to precipitate BiP and what is associated with it. Elute pellet with BiP-release buffer (Mg + ATP) and reload supernatant on a gel and test with GFP antibodies (i.e. made in mouse).
2) the same thing but starting with GFP antibodies, eluting, and then testing supernatant with anti-BiP antibodies.
If your GFP fusion protein interacts stronger with BiP than the secreted GFP alone, then you have your answer.
If you confirm that it is trapped in the ER, you can try to modify the construct by putting amino acids linker between your protein of interest and the GFP. Not putting amino acids between the two moieties can be detrimental to folding.
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