Hi, I would like to express a secretory protein with 3'-GFP-tag. After transfecting the cells, I observed GFP in the cells. However, this protein never got secreted. I do not know whether it works or not? Is it something just because of N-terminal vs C-terminal issue or we can not make a secretory protein tagged with GFP?
Forgot to mention, my protein of interest has no secretory domain.
It's not april is it?......Nope.....ok then...
Do you mean you want to express a protein with a GFP tag and have it secreted from your transfected cell? Or do you mean you want to tag a natively secreted protein with GFP?
What is your expression system??
If bacterial, there are methods of directing your protein into the extracellular space (but I'm pretty sure no further!). Search for 'Which signal sequence to use for periplasmic expression in E. coli?' here in Research gate for details.
If Mammalian/baculovirus, when you say 'secretory domain' do you mean 'signal peptide'? Without a signal peptide (or internal signal sequence) the protein will not translocate into the ER lumen. If the native protein is itself secreted then there should be a signal peptide in the full length cDNA sequence. if not It should be simple enough to clone in a suitable sequence.
I think it's more a case of chosing the system most suitable to your protein (think cysteine bonds/glycosylation etc). Perhaps once the details of your question are clear other may be able to pitch in.
Other than that, it's closer to April than I thought.......
Hi Marcus,
I wish to express the GFP-tagged protein in mammalian cells.
I have two questions.
1.Is there any signal peptide?
2. How many peptide chains in your interested protein? The effect of fusion GFP with one peptide-chain protein is better.
Hi, the protein of my interest is 26 kDa, a small single chain polypeptide peptide. It has no signal peptide (according to protein data base and NCBI). It secreted (our lab confirmed this) and it has no signal peptide, I believe that it utilizes un-conventional pathway (I have not proven this).
Other than GFP-tag option, is there any alternative approach to identify the endogenous vs transfected and expressed functional protein? My intention is, express this protein and identify it transport. The cell line that I use has this protein which is secreted out, but I do not have option to track it.
The very first problem I experienced is when I transfected a plasmid having just the cDNA sequence of this protein, I was unable to find differentiate endogenous vs transfected/expressed protein. If I tagged the cDNA with C-terminal GFP sequence, I found lot of GFP expression and fusion protein by western (~ 56 kDa) but this fusion protein never secreted out!! Whether the GFP tag prevents secretion or simply the over-expressed protein never secreted out.. I do not know.
Any suggestions to identify the unconventional protein pathways are helpful.
How can you tell for certain the protein wasn't simply released by cell death? Have you tried using an inhibitor of exocytosis (tetanus toxin perhaps) and see if this has an effect on the levels of protein you find in the media?
If you are using western blot as your method of detection there are plenty of smaller tags to use than GFP, ones (possibly) less likely to hinder the secretion of the protein whatever the mechanism.
If you don't have the option to track the protein without the GFP how do you know for certain that this protein is secreted. All things being equal and the simplest explanation being the most likely, you've shown that the expressed protein remains within the cell......perhaps that is the truth!
Dear colleague,
could you find an answer for your question? could you eventually detect your EGFP-tagged protein in the medium?
Regards,
Javi
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