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As part of an experiment that I am preparing, it would seem necessary for me to tag a restriction enzyme (HaeIII to be exact) with GFP. I began researching its domains to determine whether to tag the GFP to the N- or C-Terminus, but I was unable to find any previous studies where this restriction enzyme was tagged. As I got asking around, it would seem that no one has ever tagged any restriction enzyme at all.

It appears that this is only the case with restriction enzymes, because I very quickly found several articles where the HaeIII methyltransferase was tagged with GFP.

So, does anyone here know if a restriction enzyme has ever been tagged before? Is there something that I am overlooking that would inhibit the tagging of a restriction enzyme?

Thank you.

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  • $\begingroup$ I don't see a reason why it should be generally impossible to GFP-tag a restriction enzyme. I suspect, that simply nobody has had the need to do so until now. I suggest you simply try both N- and C- terminal variants simultaneously (including a flexible linker) and test their activity. $\endgroup$
    – Thawn
    Jun 8, 2016 at 9:11
  • $\begingroup$ Just out of curiosity, why are you tagging a restriction enzyme anyway? $\endgroup$
    – MattDMo
    Jun 8, 2016 at 14:22
  • $\begingroup$ @Thawn, I had the same thoughts. I'll just make both constructs and see which works better. I was just wondering if anyone has ever done it before. Like I said, I found several articles that tagged GFP to the N-terminus of HaeIII methyltransferase, so I was able to make one construct that worked well. I was hoping to do the same with the restriction enzyme, but it's alright, it won't be too much of a hassle to make both. $\endgroup$
    – CDB
    Jun 8, 2016 at 19:49
  • $\begingroup$ @MattDMo, just to make sure the cells are transformed properly. Of course, I need to introduce the methyltransferase before the restriction enzyme, so I need both to be tagged with a fluorescent protein to select whichever cells express both. Additionally, we will be observing the concentrations of both the methyltransferase and the restriction enzyme in different parts of the cell, so fluorescent tags are a win win. $\endgroup$
    – CDB
    Jun 8, 2016 at 20:05

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