It's an uncharacterized protein, and before going for protein expression and purification, we want to know the subcellular localization of the protein for gaining better understanding by generating a GFP-fusion protein.
Could you give us an idea of what kind of organism you're working on? That would probably help a lot with specific suggestions.
Regardless, I would run the predicted amino acid sequence through SignalP (https://services.healthtech.dtu.dk/services/SignalP-6.0/) to see if it has a predicted signal peptide since the Sec pathway is universally conserved and it's relatively easy to predict from an amino acid sequence. If it does have a secretory signal, you would at the very least need to locate the GFP to the C terminus of the protein since most secretory signal peptides are located at the very N-terminus. (Also in that case you would probably need to verify if your organism even can successfully secrete GFP-tagged proteins).
Thank you for answering, could you tell me the software were I can predict the Signal peptide domain, if any. As of now, I know that my protein has no conserved domains.
There is no way your protein has 0 conserved domains. Did you try SignalP or Alpha-fold or even NCBI annotations?
You can go "let's just try both" and make a C-terminal and an N-terminal fusion version.
Honestly, you should chat with your advisor.
- Badges
- Science topic