We are trying to remove a His-tagged fusion tag from the target protein by on-column TEV protease digestion during Ni-NTA purification, but the efficiency was quite low compared to the digestion during dialysis, which requires a second Ni-NTA purification step to remove the His-tagged fusion tag. Are there tricks that can help improve the on-column digestion efficiency?
Hi,
If your TEV is also his.tagged its mobility will be reduced due to binding to the column and essentially reducing accessibility of substrate to TEV, you may need more TEV or longer incubation.
I prefer to elute, column desalt (to reduce/remove imidazole that TEV really does not like!), digest in solution and perform a reverse IMAC (recapture his-TEV and your tag while cleaved protein passes through). It really is not so much more work if you have a desalt column available and much more efficient.
Hi,
If your TEV is also his.tagged its mobility will be reduced due to binding to the column and essentially reducing accessibility of substrate to TEV, you may need more TEV or longer incubation.
I prefer to elute, column desalt (to reduce/remove imidazole that TEV really does not like!), digest in solution and perform a reverse IMAC (recapture his-TEV and your tag while cleaved protein passes through). It really is not so much more work if you have a desalt column available and much more efficient.
hi shang, usually the on-column cleavage is expected to be less efficient than the batch-method, and this applies to several enzymes (it also tends to be immobilized on the resin by the common tag used for simultaneous depletion). and TEV is neither one of the more efficient proteases... once verified that the reaction buffer is ok (fresh reductant, needed pH, ...), I think the only way is to increase the enzyme amount.
For an example of on-column cleavage by immobilized TEV protease, you can see Puhl et al. (2009)
One trick if you insist on doing on column digestion, is to change the buffer of the column to something that is suitable for TEV. I don't remember, however, the buffer that TEV likes, but as Nick has said, the imidazole is not good for TEV, and it is best to not do on column digestion. You don't have to dialyze to change the buffer. You can do a desalting spin column which takes minutes to perform and is quite easy.
if the imidazole should be the problem of the on-column cut, it should be enough to perform the last column wash in a buffer without imidazole, like 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA and 1-2mM DTT (that's indicated for TEV), with or without NaCl depending on the stability of your protein in low or high salt.
Source of another cheap nuclease is here: http://www.protean.cz/en/recombinant-protein/11/endonuclease-s-marcescens
Not sure if this helps. We have seen that C3 protease has the highest on column digestion efficiency. Unlike TEV its compatible with variety of different buffers, additives and detergents used for membrane protein purification.
His tagged 3C protease is much more efficient, despite having a common his tag. The common tag is great as with an ON digestion, most of the 3C protease is also immobilised and removed. Afterward, a gel filtration column with Ni2+ pre-column easily removes the remaining protease. Or if you don't want to go through having to run a gel filtration column, just apply the supernatant to another Ni-NTA column to remove remaining His-3C protease. To ensure complete removal, just do a western using an anti His antibody to ensure the protease is gone and that indeed all of the original fusion protein has been digested.
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