It might, of course, but the His-tag can be removed if you design the protein accordingly and remove the tag with a protease.

I agree with Massimo Sandal; the His-tag could upset the overall folding of your protein if it is attached to a relevant region of you protein. For example, if the Tag is attached to residues that are part of the hydrophobic core of the protein it will probably upset the structure of your protein and it will reduce its stability and function. To make sure that your protein is happy you could increase the distance between the tag and the protein and possibly include a protease cleavage site between the protein and the tag (or you could use a different vector that already carries a cleavable His-tag).

If there are no structural predispositions for the detrimental effect (like N-terminus being part of the active site or the structural fold), then it can be only checked experimentally. If the N-terminus is disordered and exposed to the solvent, then His-tag normally shouldn't deteriorate protein properties but there are many exceptions, of cause. You can also try both termini.

Yes it may ! See attached paper

Article Dimerization and DNA binding of the Salmonella enterica PhoP...

It depends on the proteins to proteins. Most often it do not effect the structure of your proteins in solution. If you want to do only functional studies then you dont need to remove the tag, but if you aimed to crystallize it then it will be good to remove the tag.

Yes, although a his tag can simplify your purification, it can influence the structure as well as the function of your protein. Unfortunately, you won't be able to predict this in advance. At the end, you must analyse whether yout his-tagged protein remains functionally and structurally intact (check the activity and stability of your his-tagged protein with respect to the untagged)

At others have said, it may depending on the nature of the protein. I have observed this for one of the proteins I have worked with. Interestingly the his tagged enzme had higher activity than the not tagged one. If you are worried about having a large charge in one point of the protein, you might reconsider alternating histidines with glutamic acid residues, or consider using HAT tag from Clonetech. Or just cut it off as it was suggested.

I would go ahead with His tag, if your activity is not according to your expectations then you could cleave it. It also depends on how many residues we are talking about including His-tag, protease site, and other extra residues. Also see this paper "His-tag impact on structure". Crystallographers generally dont like extra flexible residues. If you see structural genomics papers they never remove His-tags. Difficult to generalize and need to approach in protein-specific way.

As everyone has said, you'll just need to test this empirically.

Most likely the his-tag won't have any effect but you have a 7 Kda protein and a 6His tag will make up 12% of the molecular mass of that so it is wise to test the function thoroughly against native protein.

As for introducing protease cut sites, you need to be careful as you'll be adding another set of 'foreign' amino acids to the protein. In my experience protease cut sites sometimes have worse effects on the structure/function than the his tag - but maybe that's just bad luck! Also ensure the site isn't present on the other parts of your protein or you'll end up with fragmented protein.

Often the His-tag rather than affect the activity of the enzyme can affect its stability as it may promote its aggregation and subsequent precipitation.

The answer is "maybe". Often though, in terms of specific activities and kinetics, researchers can get away with having a His-tag on their protein. The only answer is you will have to express both untagged and your tagged protein. Check your specific activity in the lysate as well as run protein expression profiles. If the N-terminal tagged protein has activity, purify it and check for activity again.

If the tagged protein does not have activity in the lysate, two good solutions are check your expression conditions (amount of Induction, temperature and media (try TM media)). If not, you may have to try a c-terminal histag.

Thank you everyone for your suggestions and information.

Mikhail, the N-terminus is not involved with the active site and it is exposed to the solvent. I agree that it will depend on the nature of protein and as many of you have said that the his-tag should not affect the activity or structure of the protein, hope that it happens in my case. We do have kept an option of another vector which has a cleavage site for the fusion tag.

I prefer not to generalize, it is case by case, as far as I know. Some proteins, in term of structure and activity, are seriously affected by the tag, while the others are not. But, logically, If the tag is far away from the active site, it is unlikely affect the activity. In term of structure, I would say that the overall structure might not be changed, if the tag is far from the core structure. However, please note that when you are attempting to have the structure through crystallization, the flexibility of tag might causes serious problem.

The FLAG tag may be a useful alternative. As the most hydrophilic of all tags, it rarely causes activity changes because it does not tend to interact with the protein's folded structure. Furthermore, it has a net -3 charge as opposed to His6, which tends to have a slight positive charge. As others have said, it goes on a case-by-case basis, but FLAG tag is definitely useful for the great majority of proteins. Conveniently, it contains a protease cleavage site within its 8-amino acid sequence as well as an epitope for a mild-purification antibody. Regarding crystallization, both His6 and FLAG have appeared in multiple crystallography reports, so the fear that it gets in the way of crystallization is not necessarily true. In fact, by adding net -3 charge, it allows a shift in isoelectric point that might be useful, not to mention its metal-binding site.

It is difficult to predict but it is possible especially if you put the His-Tag in C terminal position. For me, considering the very small size of your target protein, it could be preferable to produce it in fusion with a larger partner (MBP or Trx for example, good proteins to increase the solubility) and to foresee a cleavage by a specific protease after purification

good luck,

Colette

not necessarely but it clearly depends on the nature of the protein. if you are considering this type of purification i s because it might really improve the time of downstream procedures. I was worknig with the same system and I had the His-tag in the C-ter. to test if there's any change in the structure / function of your prot, purify it both by this method and an alternative one (without his-tag). then analyse both function and structure. If there are no changes, then go ahead!! you'll save a bunch of time!

You can try NEB intein system for no tag protein that still allows tag purification

It depends on the protein, however you can try to put TEV protease site in between His tag and the protein of interest. After purification you can easily remove the tag with TEV and perform dialysis with 2 kDa cutoff.

It definitely affects the structure of the protein, at least the epitopes. I have purified native proteins from E.coli wit the Histag by Cobalt affinity and injectetd in rabbitts. Sometimes I get a nice antibody against the His tagged version that would not recognice the native version, also expressed in E.coli, half as well. And as you have heard before one of my proteins worked nicely with an amiho terminal his tag, but the carboxy terminal tagged version would not work.

Rajeev, Its a simple but hard to give right answer. Every one has answered your question and they right as well. If I were you, I would make His6/His8-3C-protein. pET20b or 22b doesn't have C-terminal his tag. So you have to do more work or choose vector with N-terminal his tag like pET28a (Which is my favorite and also has Kan+). Make some protein and cleave half of it and do one more round purification to remove uncut protein and the tag. Compare them with assay you have. You have the answer.

Good luck.

My experience with His-tags is that they can "stabilize" a protein structure. In the case of human topoisomerase 1 with a N-terminal truncation; the His-tagged N-term. truncated enzyme had similar activity as the wild-type enzyme, however, after removal/cleavage of the His-tag the enzyme lost activity and processivity. This result was supported by another N-terminally truncated version that did not have a His-tag.

So the solvent exposed His-tag was interacting with the globular domain of the enzyme.

Yogesha, right now we do not have pET28 system, however we do have pET32a which has a N-term His-tag and can be cleaved. We are working with this system too.

Gary, its interesting that His-tag stabilizes the protein and retains its activity. Lets see what happens in our case.

The issues to consider are assembly state, stability, metal ion content, conformation, and activity. Regarding the His-tag as stabilizing truncated proteins, we’ve certainly seen some cases of this in different systems. If you are working with a truncated protein, then a tag replacing the N- or C-terminal truncated part may stabilize the expressed construct by partially covering an otherwise exposed surface that is not exposed in the intact protein. The His-tag will likely be flexible (as noted by others) so it may cover an exposed area on the protein surface that in your case (which is not a truncation) could be an interaction or assembly site. As a general rule, it’s worth the time investment to check the assembly state of your protein with and without the His-tag, i.e. changing the surface may alter the assembly. Small proteins may be monomers but are more often than not multimers. In a 50-protein test set, small angle x-ray scattering showed that 60% were multimeric (see GL Hura et al. Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS) Nature Methods 6:606-612, 2009). So your His-tag could impact the assembly state as well as the conformation or activity of the protein. It’s better to check for this than waste months on a misassembled protein. A remaining point is that Nickel columns are not only good at oxidizing Cys from Zn ligands to disulfides (e.g. EMBO J. 1999 18, 6260-70), but also may compete for and remove metal ions so if you have a possible metalloprotein check for metal content after purification.

And if you are going to use a protease to remove the His-tag, I would check your protein sequence for the protease recognition site (not including the one between the His-tag and the protein) as well as for any cryptic sites that many of the commercial proteases have.

Hi Rajeev!! How you doing..Regarding your question i can say that in our lab also we frequently use PET28a, PET23a, PET41a etc..which has His tag in C terminal and N terminal..we clone our gene and get the protein. After purification we use it for different activity and interaction studies..His tag never effect us in any type of activity..further we crystallized two of our His tag protein..and now a days solving that crystal..so finally i can say that you don't need worry about the presence of His tag in your protein..

All the best for your work..take care.

Here is my 2 cents: You might consider to try SUMO-His tag expression vector (Enzymax). You can use His tag for purification and SUMO tag for fusion tag cleavage by Ulp1 protease. Here is why:

1. Enhance protein expression and solubility

2. More efficient and cost effective protease comparing to Thrombin, TEV, and EK, and only need 1ul (12U) to remove fusion tags from 1mg purified protein with >95% cleavage at 4*C for 16 hours.

Ulp1 is a highly active SUMO protease that specifically recognizes the tertiary structure of ubiquitin-like protein, SUMO, rather than a short sequence as other commonly used proteases such as Thrombin, TEV, and EK. After digestion, the His-tagged Ulp1 can be removed by His tag affinity chromatography.

For more details:

http://www.enzymax.net/sumo%20protease.htm

Yes His-tag alter secondary conformations and affect activity too. But still i do it, as it is likely to give stability to proteins