Absolutely. Here are two examples in which the tag does indeed influence the activity of the enzyme. I worked on one of the paper (the one about HLADH and His-HLADH and we observed a slight change in pH optimum after the addition of the His-tag. The other paper is also related to this topic (even though not specific for the pH optima change) and i found it really interesting when i was wondering about the effect of the tag on enzymes. We still don't know what caused the change in pH optimum for HLADH, but interestingly, after immobilising His-HLADH onto a solid support, we observed a further shift in the pH optimum towards the initial value (of the non tagged enzyme). This last paper is in press and will be available soon, should be interested. I hope this helps a bit.

Article Unexpected influence of a C-terminal His-tag on the processi...

HI Boopathi

Although N-ter His tag is very commonly used for purification of recombinant proteins and they work fine....but the concern raised by you is also very likely...so you can cleave the His tag and analze the properties again...

bye

Some times, tag plays crucial role and alters protein's actual behaviour. For example if the N terimnus of a protein is responsible for interaction, the tagged version may hinder the molecular interaction! this is the reason structural biologist try to solve the protein structure maximum with cleaved versions. So please trust the data what you have without your tag!

@ Melissa you have explained ceratian possible inetractions due to tags very well....this should be really helpful to Boopathi..

@ Melissa Call, the enzyme is from a hyperthermophilic organism and the purification of protein involves a heat inactivation step. So the contaminants from the host cells can be ruled out and also the temperature used for the enzyme assay is above 70C.

What I believe is that the substrate specificity changes can be due to the changes in active site geometry if the His-tag is present close to the active site. But the shift in pH optimum should be related to changes in structural integrity of the enzyme. The presence of His-tag did not perturb the temperature optimum and the stability of the enzyme. The divalent metal ions had no effect on the activity of the His-tagged enzyme upto 10mM. I haven't used the His-tag cleaved enzyme so for, so I need to cleave the tag first and do the assay to compare.

Boopathi, I agree that the His tag is likely close to the active site, which I would think could also change the pH optimum. The glucosidase is functioning through an acid-base mechanism, and His residues are well known to function as proton donor-acceptors in such reactions. Certainly assay without the His tag should give you a good idea.

What is the oligomerization state of your protein? Sometimes, if the N-terminus is involved in/important for oligomerization, the tag can result in a monomeric state that is different in characters/activity to the oligomeric form(s).

Optimum pH may be assay-dependant. pH studies are really hard to do. In this case, the presence of ionizable groups from the His Tag, if they are close to the active site, may act as "phantom" groups with their corresponding pKas. Take a look to that possibility.

Hi Boopathi.

We have been working with a small acidic biomineralization protein (6.4 kDa) in our lab where it is thought that the C-terminus is the active portion of the protein. However, we have found that the His6 N-terminal tagged version of the protein was not as effective at biotemplating the nanoparticles as the untagged counterpart.

This may be due to the His6 tag chelating with some of the metal ions in the solution, thus preventing them from being concetrated by the active areas on the C-terminal section during mineralization. However, the His6 tag also significantly alters the pI of the protien from ~4.5 to ~6.9, this could also alter the activity of the protien. Unfortunately, as there is not a solved diffraction structure for our protein, it is difficult to know if the tag may also alter the strcuture of the protien, but some preliminary models from ab-initio folding indicate that the presence of the N-terminal His6 tag doe alter the structure of the protein.

I would say that the presence of the tag may well have caused the differences you have seen in your protein, but as I don't know much about the size and activity of your protein, it is difficult to be sure. I think cleaving the tag is an excellent idea, as working with the enzyme as close as possible to it's natural state will probably give you more useful information.

Good luck!

Absolutely. Here are two examples in which the tag does indeed influence the activity of the enzyme. I worked on one of the paper (the one about HLADH and His-HLADH and we observed a slight change in pH optimum after the addition of the His-tag. The other paper is also related to this topic (even though not specific for the pH optima change) and i found it really interesting when i was wondering about the effect of the tag on enzymes. We still don't know what caused the change in pH optimum for HLADH, but interestingly, after immobilising His-HLADH onto a solid support, we observed a further shift in the pH optimum towards the initial value (of the non tagged enzyme). This last paper is in press and will be available soon, should be interested. I hope this helps a bit.

Article Unexpected influence of a C-terminal His-tag on the processi...

Sorry before one of the two papers was not properly attached. Here it is

Article Horse Liver Alcohol Dehydrogenase: New Perspectives for an Old Enzyme

Dear Boopathi,

the answer depends on the structure of your protein.

If the N-Terminus is involved in catalysis or oligomerisation, changes are very likely.

Since the structure of many B-Glycosidases are known, you can try to find this out easily by looking in the pdb.

e.g. 3GFX Crystal structure of acid-beta-glucosidase with isofagomine at neutral pH

This structure shows an well ordered N-terminus close to the active site and N-terminal residues are annotated as functional for binding.

Homolgy search may indicate, which structure is most closely related to your protein and which features you can expect.

Marco

@Refaat, Marco, the protein is in dimer form. There is only a disulfide bond between the two as inferred from the homology modelling of the structure. The molecular weight of the monomeric form of protein was 57kDa.

@Melissa and Daniela, thank you for sending the papers. It was really useful.

Dear Boopathi,

Danielas papers already made a strong point for the potential influence of tags on the properties of enzymes. Please find attached a poster with another set of experiments giving further support for even small tags having signficant effects, in this case on the Km ATP of a protein kinase and its sensitivity to small molecule inhibitors.

So a real careful comparison of your protein of interest with tags either N- or C-terminal or with the tags removed might be very helpful indeed.

Daniel

Hi Boophati,

as many others have pointed out, a his-tag CAN have a big effect on enzyme efficiency or specificity, even though tthe well-documented cases are few.

I just signal a further couple of old references in which a His-Tag was reported to make a difference in terms of catalytic behavior.

Gross et al (2003) Antimicrob Agents Chemother. 47(3):1037-46.

Walker et al. (2001) J Biol Chem.276(24):21343-50

Although is not at the N-terminal but at the carboxy terminal of our enzyme, we have developed an artificial his-tag metal binding site which is capable to switch “on” and “off” our enzyme by addition/removal of zinc (Catalano-Dupuy et al, 2006, Biochemistry 45, 13899). It is a good example of the profound impact that a small his-tag may introduce to the properties of a protein.

Tags can always influence protein function. Consider putting them at termini that are less likely to be important. Otherwise plug in the middle in a non-conserved region.