Hi
I used Ni column to purify my his-tagged protein (57 kd), after that, I cleavaged it with TEV protease that gave me 47kd for His-tagged, 52kd un-cleavage proteins and 12 kd my target protein
now I want to use gel filtration as last purification step after cleaving the protein, can I add my sample which contains TEV protease to gel filtration or that may affect the separation
thankssss
57 kDa his tagged protein
45 kDa protein after removing his tag
27 kDa TEV protease
12 kDa his tag
You can directly apply the sample containing TEV protease to gel filtration without any problem.
What is the size of the tev protease? If it is not close to your target protein you will be fine. Anyway you will need a large column to separate your mixture.
Hello,
Most commonly commercially available TEV is His-tagged as well. This is useful for subsequent removal of the uncleaved protein and TEV with another run of Ni-column - your cleaved protein will not bind as it will have His-tag removed while any uncut protein will bind and TEV will bind as well (if your TEV is His-tagged). After that it is advisable to run gel-filtration. TEV protease will run as a 27-30kD protein.
You can run gel filtration column directly after cleavage with TEV only if the sizes of cleaved and uncleaved proteins and TEV itself are different enough so you can achieve good separation.
JJ
Hi,
Technically you can purify your protein just with one Ni and one gel filtration column. You do not need to run the second Ni column because of difference in size of TEV protease and your target protein (27-30Kda vs 12Kda). Just gel filtration column followed your Ni column should be enough.
When you load your whole construct (which consists of a Tag, a fusion protein, a TEV recognition site and your targeted protein) to the column, you have to make sure that you fully loaded (or at least 90%) your column. Then you wash your column with 5% of your eluting imidazole solution. This way you will remove all non-specifically bound protein from the column. After that you load your TEV protease. Because it usually has a His Tag, part of it will bind to your column if it was not fully loaded. And that is amount of TEV you will lose (not doing it job – cleaving your protein). If you have sophisticated equipment, you can make a close loop and circulate your eluted solution through the same Ni column again overnight. You have to know, that TEV protease has lower rate than serine proteases, it is a sequence-specific cysteine protease. So, keeping overnight will increase the yield of your target protein. After that you run your solution, which should contain mostly His-tagged TEV protease and your target protein though a gel filtration column. Because of difference in size, your protein and His-tagged TEV will be well separated.
Finally, you clean your column with high concentration of imidazole buffer and then recharge your Ni column.
But in general, for other cases, when your target protein is comparable in size with TEV protease, I would recommend as Janusz suggested to run a second Ni column, to separate His-tagged TEV protease from the target protein.
George.
thanks a lot
my target protein 12 kd and TEV protease 27 kd , so I hope there is no problem if I run Gel filtration if I run second Ni column may I lose more from target protein
if I run second Ni column may I lose more from the target protein
Hi,
You do not need to run a second Ni column. Your target protein is much smaller than any other protein in your yield solution. So, it should be a nice peak after a gel filtration column.
George.
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