Im working with a His-Tagged protein that aggregates (amyloid like) on nervous system neurons and I need to evaluate the kinetics of this process. Do I need to remove the His-Tag of the protein?
The eternal question.
In the distant past, I ran into problems that results were not reproducible, when performing an enzyme assay with his-tag or after removing the his-tag.
In the end, I decided to purify the protein without his-tag and found to my delight that the enzyme activity was suddenly many-fold higher and the enzyme kinetics much simpler and easier to explain.
From then on, unless it was for a screening just with SDS-PAGE or similar, I never used the his-tag again.
If the N or C-terminal of your enzyme is in any way involved in the enzyme activity, the his-tag will have an effect, and if it is only slowing down the agility of the enzyme's terminal part, due to the added mass.
The Correct thing to do is to test whether the his-tag has an effect by doing your aggregation experiments with and without the tag.
In some cases there is no difference others there are large ones. If there is a difference that tells you that histidines are doing something and I would look into metal effects, the effect of imidazole etc.
If your construct has a protease site after your his-tag run your IMAC and use a protease that is either his-tagged or conjugated to gel beads. Then dilute your sample to lower the imidazole content and run it on the IMAC column again. your run through should be your protein.
The answer is in the question. You are evaluating the kinetics of the process of aggregation for your native protein, not the his-tagged one. To be sure about the influence of the His-tag, you need to compare it to the untagged protein. Remember that most His-tag contructs that can be cleaved off by a protease leave additional amino acids on you protein which can also have an influence.
So the best option would be a construct where you can get the native N-terminus free of additional amino acids. Purifying untagged protein is more difficult, so you could use e.g. the His-SUMO fusion tag which can be cleaved by its protease.
Hüseyin Besir protein purification is not that difficult. The problem is that it is not taught anymore in universities or only the basic theory or only the very basic tuff, like gel filtration.
As mentioned in my previous post, I went away from using tags, because it affected the proteins activity. Unless you can remove all residues ,as you describe, it is never clear, what affect the remaining additions can have on the protein. The push for me to get away from tags for real came for me, when in one case it became clear that a single remaining residue was interfering.
I didn't mean absolutely difficult, but compared to tag-based purification, it is more difficult. Plus, it depends on the protein. You're right, the way that protein expression and purification is taught nowadays it's unlikely that purification of untagged proteins will become the first choice for most people.
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