It depends on which resistance protein you are fusing to and what the N-terminal tail is, but in general it should work. There is an extensive literature of fusions with beta-lactamase (but it needs a signal sequence at its N-terminus). CAT fusions have also been used.

It should be working.

Make sure everything is in frame.

If it's in frame and the protein fold correct, it should work...

Hi

it is better you place antibiotic gene under a separated promoter. This cause antibiotic protein have any effect on folding of purpose protein.

It depends on which resistance protein you are fusing to and what the N-terminal tail is, but in general it should work. There is an extensive literature of fusions with beta-lactamase (but it needs a signal sequence at its N-terminus). CAT fusions have also been used.

We have an positive experience with ermB gene, just keep everything in frame.

For the target protein of interest, where in the cell is it (putatively) localized? In the cytoplasm or in the periplasm? This will affect your final choice of antibiotic resistance protein that you use. Definitely, your reporter enzyme needs to be in-frame with your truncated construct. As mentioned by Michael above, the two main antibiotic resistance reporters that have been used are beta-lactamases and chloramphenicol acetyltransfereases (CATs).

Beta-lactamases target the enzymes responsible for cell-wall (peptidoglycan) biosynthesis in the periplasm; as such, they only provide resistance when localized to the periplasm. So, if your truncated protein of interest is a cytoplasmic protein, and you express it with a beta-lactamase reporter, your cells will still be susceptible to beta-lactamase drugs in the media.

Chloramphenicol acetyletransferase (CAT) is only functional when expressed in the cytoplasm, as it requires acetyl coenzyme A (also present in the cytoplasm) for the inactivation of chloramphenicol via this mechanism. As such, a CAT fusion expressed in the periplasm will not confer drug resistance.

(If you're not sure as to where your protein localizes, and you simply want to see if it is expressed or not, you could create a fusion to either mCherry, or superfolder GFP; both are able to fluoresce in either the periplasm and cytoplasm).

Hi,

we have successfully fused Km resistance gene (3'-APH) with red fluorescent protein (mRFP). This is absolutely active protein (both domains are) and we don't see any difference in activity compare to WT 3'-APH.

If you are asking about bacterial resistance, all these above answers would help you. For mammalian resistance markers, you can try puromycin fusion protein.

Fusing two independent proteins could affect the function of either protein if the resulting fusion protein would cause either or both proteins to be inaccessible for translation. Perhaps it would be not a bad idea to check for the protein structure of the fusion protein to determine this point. Another point to consider is glycosylation, if the resistance protein is fused with a protein with a glycosylation site then it might cause a chance in function.