I was wondering whether you can express the following:
CMV Promoter - NeomycinResistance - viral2a - mitochondrial signal peptide - GFP - Terminator.
In theory the 2a will self-cleave and you get a NeoR protein and a mitochondrial tagged GFP.
However, the T2a ("viral 2a") leaves a proline attached to the N-Terminus.
Will this mess up the signal peptide, so the GFP will stay in the cytosol? Or will it still work and be imported into the mitochondria?
The same question is asked for chloroplast targeting peptides, ER localizaton signals, and extracellular secretion signals.
Hi Andreas, I am no expert, but I got some information from De Felipe, Biotechnol J, 2010 (doi: 10.1002/biot.200900134) when I was interested in a similar question. Maybe you can find some answers there. If I am not mistaken, what you are proposing should work... Good luck!
Hi, we also put the signal peptide in the first protein (CFP) (as Andrei suggest) and the targeting was as expected (no problem with the extra proline). The second one after the 2A peptide worked properly in the cytosol. You can have a look in the publicationd. Good luck
Thanks a lot for your replies!
Well, of course in this case you could switch it. I choose a bad example.
But if I tagged GFP to the mitochondria and RFP to the Golgi (retention signal).
Or two proteins into the mitochondria.
MLS-GFP -- T2A -- MLS-GOI
so I get a correct MLS-GFP, but a proline tagged GOI.
If I tag more GOIs into the mitochondria in tandem...
I had the same question and found this Cold Spring Harbor protocol/review of 2A peptides, which comments that 2A generally does not affect the localization of downstream proteins except when the upstream protein is targeted to the ER lumen.
Design and Construction of 2A Peptide-Linked Multicistronic Vectors
Andrea L. Szymczak-Workman, Kate M. Vignali, and Dario A.A. Vignali
Cold Spring Harbor Protoc; 2012; doi:10.1101/pdb.ip067876
http://cshprotocols.cshlp.org/content/2012/2/pdb.ip067876.short
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