It can be a number of things. Either you don't have enough reducing agent if the proteins has exposed cysteins, or there is a protease making chaos around.

Maybe you can try to stabilze your protein in some steps or in the end storing it with a ligand. That should make them more stable.

By that way, if the protein does not bind to the column it really does not sound like a good idea to use it as a purification step. Why don't you try a MonoS instead?

First, how is that you express this protein. Does it needs refolding?

I had a very similar problem with the enzyme I work with. In my case, the protein was expressed in its insoluble form and then refolded. I always thought that the problem was the refolding step itself. I've tested inumerous buffers but none solved the problem and I still had a enormous variation in enzymatic activity between purifications batches.

I always got a aproximated amount of protein on each purification, but because it was denaturated and renaturated, I had no control on how much of the denatured protein was able to renaturate properly. So when I used x micrograms of protein in my enzymatic assay, I didn't know how much was in active form.

What I did was to add a additional step of purification. Doing this I lose some of the protein in the process, but it is worth it. After the refolding I centrifuge my sample and I use the supernatant, even if I can't see any precipitate. Doing this I believe I diminish the inactive/active ratio, diminishing my bias.

Well I don't know if it is your case, but I hope I can help.

Felipe we have tried adding DTT and EDTA and the activity does not change very much. And the reason we run it over MonoQ is because while it does not stick, all of the contaminating proteins do seem to stick.

Christiane we do not need to denature our protein. However, I do believe that the same sort of issue is occurring with only some of the protein being used in the assay being active, but I believe it is due to aggregation (it runs as a huge mass on our gel filtration column) but we cannot seem to be able to control this problem at all.

Thanks for your help!

Did you make glycerol stocks of your expression system the first time and keep using those?

If yes, try transforming the plasmid freshly for each batch. I never did that although recommended by the competent cell vendor until I finally ran into a protein where it was actually necessary. Don't ask me why fresh transformation is sometimes required, but it works in a few cases!

S.

Thought about whether it might be a problem with inclusion bodies? These are aggregated misfolded proteins. You can look for them in the pellet of your lysate. You might have success with a significantly reduced growth temperature. For our protein we have found that 4 C over a week (or 18 C over a few days if in a hurry) is by far the best way to get properly folded protein levels. Even a slight misfold is enough to destroy activity even though the protein looks much the same on a gel or FPLC. So you end up with a slightly misfolded but still soluble protein that is inactive. Very hard to purifiy away.

You also might consider a centrifugal filter to concentrate between your IMAC and MonoQ steps, as I often find that proteins are very much more stable when prevented from being diluted.

Also with regards to stefan's comment on glycerol stocks. If your cells are kept at -80 C in a glycerol stock that is never allowed to thaw (keep on dry ice when using) then they should retain the plasmid and health for a long time in MOST cases. If this is not the case then you will have trouble with plasmid rejection and other processes that cause loss of expression.

Is your DTT solution (or DTE) prepared fresh daily (It can oxidize readily, within a day or few, especially at RT)? Or as fresh as possible? What concentrations of DTT, EDTA employed? If aggregation is a suspected problem, here are some web suggestions.

Additives may prevent aggregation. Try glycerol, 1-2% ethylene glycol, 1-5% polyethylene glycol (PEG), high salt, low salt, different pH (may be too close to pI), some detergents (0.1- 1% Chaps, Triton X-100, Brij-35, Tween 20, NP-40 or other). Using detergent screen from Hampton and then monitoring through DLS could be a good way of screening.

When His-tag protein is purified through Ni-NTA column, during elution Ni may get eluted along with the protein and binding of proteins to the eluted Ni could be the cause of aggregates. Try dialysing the protein against 10 mM EDTA or addition of EDTA to the tubes used to collect fractions.

Use an alternative column for purification of His-tag protein: Cobalt-TALON. Ions on the column will not detach because it has one more dental holding the ion.

Try using low pH to elute His-tag protein from the Ni-NTA column. It is also noted that a longer His-tag reduces protein solubility and favours aggregation.

It has been observed that b-mercaptoethanol can bind to the cysteine residues and form a covalent adduct. DTT may be your best bet.

My suggestion is to first, follow the specific activity of the protein during purification from beginning, ie cell extract, to the end. The specific activity is defined as units of turnover by the enzyme per unit time divided by total mg of protein. So you also follow total protein. This will allow you to troubleshoot as to where your problem(s) lie. Second, enzymes are stabilized by their substrates and cofactors and possibly products, so I would try adding arginine, methyl arginine, SAM to the buffer during the purification, or whatever your substrate is. This will help with the aggregation. You may also want to supplement the growing of the cells, whatever they are, with SAM and any other cofactors it uses. I wasn't sure if it has any metal requirements. I thought methyltransferases do have this. If it turns out that your activity is lost after the NI column, then maybe you are replacing Ni for another metal ion. This could explain the variability, ie if your resin has varying amounts of Ni that exchange with your cofactor.

S Anotnonysamy, 2012 said their methyltransferases aggregated. They expressed with MEP50. So I express or purify with a substrate synthetic, or otherwise, and then purify. Methylarginine might work at mM concentrations perhaps.