I am producing two proteins with E. Coli. One is fused to a his-tag, the other one to a Thioredoxin tag. Both can be cleaved off with TEV-protease. When I run a gel after purification, I can detect both my proteins, but also a third protein of unknown origin. This means however, that it is somehow his-tagged and the tag also gets cleaved off by TEV-protease.
Do you have any suggestions as to what the reasons for this are?
There are a couple of proteins that stick to Ni-NTA and can co-purify. Some known proteins are YodA, Hfq and FKBP. For YodA co-purification please see Tian et al 2005, Meth. Enzymol. 394, 321-334.
Have you checked if it might be your TEV-protease that still stays in your mix?
Maybe you found a protein that strongly interacts with your protein(s). If you are in the research field it might be interesting to find out, what protein it is, what its function is, if protein 1 or protein 2 is the interacting partner and so on. Might get pretty interesting scientifically.
If you are producing the two proteins for a commercial purpose or need clean proteins for a different reason, you might get rid of the interaction by changing the pH, detergent, reducing agents and so on... But be careful not to destroy your proteins of interest as well.
Good luck!
I agree with Vivica. The most possible reason for your results is that you are detecting your TEV protease on your gel. You will have to perform a FPLC step in order to further purify your proteins.
An additional idea: If you want to check if the third protein has a His-Tag you might blot your gel and incubate with an anti-His-antibody.
Another aditional idea: better and quicker than what Vivica proposed, you can run a batch with His-tag purification resin and see on what fraction do you obtain your protein.
The extra protein could be something that is binding to the resin and not the His-tag. That should be easily taken care of by running the prep through a resin-only column before the His binding column.
What size is the unknown protein? It is very common to see the TEV post cleavage depending on how much TEV you use and how/whether you clean up the proteins. Are you removing the TEV post cleavage?
There are a couple of proteins that stick to Ni-NTA and can co-purify. Some known proteins are YodA, Hfq and FKBP. For YodA co-purification please see Tian et al 2005, Meth. Enzymol. 394, 321-334.
I agree with Mario and Vivica. There is a big chance that some non-specific protein binds to your resin (I found it few times when I was preparing Western blot after purification). I think also that if would be easier for us to suggest something if you tell us the molecular mass of this proteins, both expected and unwanted. And maybe try to do WB after purification but before using TEV.
Good luck :)
Hi guys,
sorry for the delay...
Forgot to mention the proteins size. It is around 37 kDa. So the proteins Mario named are too small. Furthermore, TEV protease gives rise to another band at around 27 kDa.
I have two lanes:
1. elution of the column --> two target proteins, plus unkown protein.
2. flow through of the column after incubation with TEV protease over night --> two target proteins plus unkown protein (TEV protease binds to Ni-NTA and is eluted afterwards).
So I think unspecific binding to the resin is unlikely, because it would only appear in the elution or the flow through fractions. Since it seems to co-purify with my target proteins, it really might be that I observe some kind of interaction. However, since the target proteins are of human origin, this is probably just random?
Hi Michael, I'm a bit confused about your description:
"2. flow through of the column after incubation with TEV protease over night --> two target proteins plus unkown protein (TEV protease binds to Ni-NTA and is eluted afterwards)."
You took the flow-through of the Ni-NTA column (e.g. material that does not bind Ni-NTA) and incubated that with TEV?
Or do you mean that you took the eluted material, incubated that with TEV, re-applied that to Ni-NTA and collected the flow-through and ran that in lane 2?
Hi Steingrimur,
the latter version is correct.
I ran two purification steps. In the first I collected the elution of the column, so that I only had protein with a His-tag (or unspecified binding). Then TEV-protease was added which cleaved of the tags of the target proteins. Lastly, I re-applied the solution to a new column in order eliminate the tags and TEV-protease. Hence, I collected the flow through.
I took a sample directly from the first elution and one from the flow through of the second column run. I ran a gel and both samples showed this third protein.
Hope this makes it a little more clear.
Hi Michael,
another thing that I had observed before is the observation of a dimer band. Although SDS should denature all proteins, some folds are so stable that they just do not denature completely and if it is a tight dimer a band roughly at the size of a dimer occurs (depends on shape). Cutting out the band, digestion and MS/MS would be able to tell you which protein it is. I am curious what it is.
Hi Michael, thanks for the clarification. It does seem that you have a foreign protein that is specifically binding the Ni-NTA.
If it is a foreign protein, then it is unlikely that it has a 6-his tag and you might be able to elute it off at lower imidazole concentrations. Maybe do a linear or step imiazole gradient.
Mario makes a good point that your protein could be forming an SDS stable complex. Do any of your proteins have a free Cys? You could try boiling the sample with DTT and see if the band goes away.
Hi
do you have control?
you can purify proteins from non-transformed bacteria that growth at the same condition and if you have that band it is a native product that bind to the Ni-NTA .
Please harvest bacteria and make lysate and do a Western blotting using his antibody.
Thank you all for the input! Absolutely gave me a better understanding which circumstances might lead to this band. Let's see what I can figure out.
@Steingrimur: I actually boiled my samples in DTT before applying them to the gels.
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