I will locate a newly induced human protein in yeast to test its location correctly. The original position of this protein in human cells is ER. So I decided to add a fuse mCherry to it, then use fluorescence imaging to detect if it can locate at ER in yeast.
The question is where should I put it? N-terminal or C-terminal? Should I use some flexible linker between them?
The decision of where to put the mCherry fusion tag would depend on the specific protein being studied and the goals of the experiment. Generally, adding a tag to either the N- or C-terminus of a protein is a common approach for subcellular localization studies. A flexible linker can be used to connect the tag and protein, which may help to minimize any potential disruption to protein function. Ultimately, the best approach would depend on the specific protein and experimental design.
To add to Rajan Singh , as an a priori test you can check for the structure of your protein in Alphafold (unless its structure is resolved by other methods) and try to guess where it would impact less the folding, the function and/or potential interactions of your protein of interest.
If you know domains that are important for the targeting, for interactions, or for the functionality of your protein, try to put the mCherry tag as far as you can of these regions.
If your protein is targeted to the ER through a known sequence that is in either N' or C', go for the other terminus since you seem to be interested in the location of this protein.
To finish, if it's a TM protein, you can consider to insert the tag within a loop between different TM segments.
I would recommend you make both versions. If they differ in localisation it can tell you something about your protein of interest. If both have the same location happy days.
Based off my experience, mCherry on the N-terminus also behaves as a chaperone and aids in the faster folding of the protein downstream. Yields are also extremely high in comparison to C-terminus mCherry. As mentioned by others, it is quite protein dependent. If you add mCherry to N-terminus and it affects the functionality of your protein, you are better off having it on the C-terminus, in spite of low yields.
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