It is in general almost impossible to predict the potential effects of tags on the biological activity of recombinant proteins just based on the position of the tag - but a short HIS-tag does only very rarely influence its protein significantly - the problem is usually the other way: The tagged protein masks the HIS-tag and the native purification does not work efficiently

1) you can use only one C-terminus for linkage plus N-terminus of the other protein, thus you will still have one N-terminus and one C-terminus available for tagging

2) it's hard to say, that depends whether is the terminus buried in structure or close to active site etc. If it's exposed freely on the surface, it should be fine.

Thanks Tomas. Yeah the its exposed freely on the surface. The difficulty is that i am using two tags, so the other tag Cmc is on the other C-terminus of the other protein. The construction is like this: MICA-Linker-ScFv-Cmc. So the only place that is free is the N-terminus of MICA. But since i only need alpha 1 and alpha 2 for its reactivity with NKG2D for in vitro and in vivo tests i think it will work, as you said. Thank you.

I don't know what's Cmc (and Google wasn't much helpful:) but theoretically you could add the His-tag also to the C-terminus

It is in general almost impossible to predict the potential effects of tags on the biological activity of recombinant proteins just based on the position of the tag - but a short HIS-tag does only very rarely influence its protein significantly - the problem is usually the other way: The tagged protein masks the HIS-tag and the native purification does not work efficiently

Thanks Daniel, if i get you correctly His-tag is so small to affect the protein it is attached to. Thanks

In addition to what Daniel said, heavy metal binding to the His-tag could have more of an effect on bioactivity than the His-tag itself.

You are correct, thats the general idea. And indeed so far we could in our lab (with some 250+ purified enzymatically active proteins) never observe a negative effect of a HIS6-tag alone on the enzymatic activity, regardless of the HIS-tag being at the N- or C-terminus. There were however numerous cases, where the native purification via HIS-tag would not work, while GST-tag purification worked perfectly well. So I would say there is a good probability that your protein will not be negatively influenced by the HIS-tag. Whether the bioactivities of your two proteins do influence each other is however a completely different questions.

Good point made by Steingrimur, in order to address that problem you should consider to add chelating agents (like EDTA) to your final protein preparation if that is compatible with your downstream applications.

Any N-terminal signal sequence involved?

Thanks Wolfganng, no signal sequence is involved

Thanks Petri, yeah i was thinking about that, that is removing the His-tag before the in vi and in vitro test. Thanks very much, i will take that into consideration.

@Tomas, Sorry Tomas, it is Cmyc and not Cmc

As Daniel Rightly said its impossible to predict other than performing the assay and knowing as most of the times the tag does not interfere.

But in case the activity is related to ionic state of the active site. Just as the His tag binds to metal ions due to their affinity this property if essential/inhibitive for bioactivity of the protein there could be effect felt. Again this depends on the distance of the his tag over 3-dimensional space from that of the active site residues. Remember in general many protein require metal ion as cofactors. In such case His tag can bind with high affinity to metal ion cofactor.

It thus depends on what sort of function the protein of interest does and its mechanism of action.

@Kannapiran, thanks very much for your contribution, well appreciated.

Occasionally tags can affect bioactivity. If you are worried then make a fusion with SUMO(which can be His tagged) and cut with SUMO protease then you should get the native N-terminus back- although you may have to work out if your protein has its N-terminal Met cleaved off in the first place!

@Louise, Thanks very much, i just googled SUMO to see how it works and i think it is a very good alternative which should be considered. Thanks you very much for your contribution.

@Veer, Thanks very much for your contribution