In my construct I have two His tags, one at N-term and another at C-term. There is a TEV site between the N-term His tag and my gene but no cleavage site for the C-term. So, do I need to add another cleavage site on C-term as I am aiming to get the crystal structure or their shouldn't be any problem as the tag is on the C-term?
You can not say his tag is good or bad for crystallization. His tag can help you in crystallization or it can prevent crystallization.
Why are you putting his tag at both end? I think that's a bad design. you can add cleavage site at C terminus but it will not be a great help as at the end, you will end uo having similar number of extra residues as his tag itself.
I haven't cloned the gene but got the construct fron addgene.
you can always re-clone it by yourself. but again you can set crystal tray as it is as long as your protein is functional. you can not say putting his tag at a particular end is safer than other. its protein specific. I have a protein where depending on its interaction partner and context, we put tag at one end or the other. its both ends are involved in its different function. if we want to test its both function at same time, we need to have internal his tag.
Dhiraj
So, is it fine if I don't cleave the C-terminal his tag and proceed for crystallisation?
Thats perfectly fine as long as its not interfering with your protein function.if you are not getting any hits, then remove it. since you already have construct, its most easy thing to try.
1. According to my experience, both ends of the His-tags can effectively prevent protein degradation, so your construction is suitable for a protein that is prone to degradation.
2. I don’t recommend adding another cleavage site at the C-terminus, because even if the protein is finally cleaved with TEV, there will be many remaining residues that may affect the protein crystallization.
3. The C-terminal His tag may help to crystallize the protein. Anyway, you can try it as soon as possible using the existing construction and purified protein. Why not try?
Good luck!
Roger Davis Thanks. I had already started working with the original construct.
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