Hi, I am attempting to co-express GFP using a bicistronic vector that contains the T2A peptide.
(CD8 alpha signal peptide - GOI - T2A - GFP)
I am concerned that, since I am using the CD8a signal peptide, which directs the expressed protein to the membrane, GFP may also be directed to the membrane, making GFP expression undetectable in the cells.
Will GFP be localized in the cytosol if I use the vector I described above?
I look forward to your kind reply.
Thank you, and I hope everything goes well for whoever reads this.
Hello
In my opinion, In your described vector (CD8 alpha signal peptide - GOI - T2A - GFP), it is expected that GFP will be localized in the cytosol rather than the membrane. This is because the T2A peptide should separate the GOI and GFP proteins during translation, allowing each to have its own proper localization signals.
However, I'd say it's always a good practice to experimentally validate the subcellular localization of your proteins. You can use fluorescence microscopy or other appropriate techniques to confirm the localization of GFP in your transfected cells.
interresting question; T2A is a stop and restart translation; as synthesis of secreted protein are made with ribosome attached to the membrane and to a pore to translocate the secreted protein for me there is some chance that your GFP is going to end in the reticulum ...
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