His-tagged proteins

[claire84]

Hi everybody,
I have some doubts about the use of his-tagged HIV-1 proteinase for an inhibition assay.
I have to test some potential inhibitors and I would like to perform a kinetic assay to obtain the percentage of inhbition and the IC50 values.
Can the presence of the tag influence my results?
Is better to purchase recombinant hiv-1 protease without tags?

Thank you

[JackBean]

of course it can. That depends on the structure of your protein. If the end on which you have your tag is far apart from the active site, it won't influence the behavior, but if it were close enough, it could interfere. But His-tag is quite small anyway. However, you can introduce also some protease-cut site to remove your tag

The best thing you could do would be isolation of the protease from original source, protease from bacteria, or whatever you isolate it from and your protease with His-tag from the bacteria. Then compare their properties and you will see, whether there is some difference.
I bet there have been already some reports about cloning this protease, have they not? Just look, how they did it and that should be fine.

[claire84]

In our lab, we do not produce recombinant protein, we purchase them for inhibition assay....
but I think I could try to use his-tagged hiv-protease, N and C termini are not closer to active site

thank you for your reply!!!