Hi I need to purify a recombinant protein (IL6 21kDa). It's been produced in E.coli BL21 with a T7 expression vector, but it does not have a tag as yet. I was thinking of adding a his-tag and purifying with nickle beads or a column and then removing the his tag with protease. But this method would still leave some extra amino acids on one end of the native protein. Is this important? Would it affect the biological function of the protein in any way?
Are there other methods of purifying recombinant proteins and how difficult are they to carry out?
I appreciate any advice you can give!
Hello,
His-tag purification is one of the most common purification methods, as you mentioned, you 'll have some extra aminoacids, you 'll need to check if they bother your protein's activity, but normally it must be fine.
A lot of people work with bigger tags, and don't even cleave them, so hopefully your tag won't bother.
There are other methods, however in general, you carry on an affinity purification step (in this case, using nickel beads to bind the His-tag, or other tags : flag, strep etc.). Afterwards, there are several methods that can be used to purify even further your protein:
Ion exchange : depending on the pi of your protein, and your buffers, you may be able to use a cation or anion exchange column
Steric exclusion chromatography (SEC) : very useful to polish your preparation and obtain a good sample.
Now, depending on your protein and your downstream applications, you may start with the affinity step, and choose to add or no another step (probably SEC).
All of these methods are pretty easy to carry out if you do not encounter any particular problem due to your protein (solubility etc.)
Interleukin 6 has two disulfide bonds and N-linked glycosylation https://www.uniprot.org/uniprot/P05231, bacterial expression may therefore not be ideal Article Recombinant Production of Human Interleukin 6 in Escherichia coli
This article describes the production from inclusion bodies, denaturation and refolding followed by ion exchange chromatography:
Article Enhancing recombinant interleukin‐6 production yield by ferm...
Others produce IL6 in yeast (secreted) and purify it by ion exchange and gel filtration chromatography
Article Production and purification of recombinant interleukin-6 sec...
Article Large-scale production, purification and bioactivity assay o...
Even with a his(6) tag, purification with imac is not straightforward. Other techniques must be applied such as ion exchange and gel filtration
Dear Ian
In the most cases the presence of a small tag as the Histidine do not affect the functional properties of the fused proteins, however each protein and protein domain has an his own history and there is not an universal answer to your question.
However as you suggest to minimize this issue you can add a protease restriction site beetween the Histidine TAG and your protein (eg TEV cleavage). In this way if required you can remove the Histag and mantain in your protein only few extraadditional residues (eg the Glycine for the TEV recognition site) and this will allow also to further improve your sample purify performing a second subctratcive IMAC step.
However also in this case, this step to succed require that your protein is stable in the protease working buffer and for the time and temperatures that are required to the specific protease to perform digestion.
For example for TEV protease generally i incubate the sample at room temperature over night.
If you are interested you can find more information about TAG and protease on my blog ProteoCool ( ProteoCool ) at PAGe 2
ProteoCool n°22 (Overview on Tools for recombinant protein expression in E.coli)
and at PAGE 3 on
ProteoCool n°13(Subctractive IMAC)
good luck
Manuele
I was wondering is there any specific reason for you to remove His tag from your protein?
His-Tag is the standard purifycation method for a reason. The small His-tag usually does not interefere with your proteins function. However, you should still confirm it if you have the chance.
The good thing with the His-Tag is that you can apply it to both termini of a protein. There is actually only one disadvantage to a His-Tag purification:
It may has the highest protein yield of all known purification methods to me, however it can lack in purity.
This trade-off is normal protein purification. Yield vs purity. But even with the His-Tag itself you can play with this balance.
Ni-NTA and the side of protein Yield
https://cube-biotech.com/us/products/protein-purification-products/his-affinity-resins-magbeads/ni-nta/
and Co-NTA on the side of the protein purity.
https://cube-biotech.com/us/products/protein-purification-products/his-affinity-resins-magbeads/co-nta/
You may apply biotin tagging and streptavidin/avidin isolation without the use of FPLC at the first step. You may use SEC or ion exchange at FPLC, just for polishing purposes as a second step if needed....There are references for biotinylated IL-6...
Thanks for all the answers , there are certainly a lot of options to look into here.
To answer the question about why I want to remove the tag, its because we are planning to examine the human IL6 with small angle neutron scattering. So I wanted to make the protein as close to native as possible. But I think using a his tag may be acceptable in this case.
Also regarding Annemaries comment, there are certainly some issues with using E.coli for recombinant IL6, but in the paper you linked:Article Recombinant Production of Human Interleukin 6 in Escherichia coli
they use chaperone plasmids/proteins to greatly increase the amount of soluble IL6 and I have used the same technique here, it seems to have worked very well. So I plan to push on with some IMAC followed by some FPLC of some description. The FPLC will be more challenging part for me as I have no experience but thankfully there are some researchers here that do.- Badges
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