Hi All,
I have used MBP tag to help solubilize my target protein. It works really well. However, whenever I cleave off the His-MBP tag and try to clean up the sample by passing through another Ni column, I found that there is always unbound His-MBP in the flowthrough fraction. Also, there are cleaved protein (without His-MBP tag) in the Ni column bound fraction. The only way to explain this would be that the MBP tag bind to my protein to help solublize it and cannot be removed after the Ni column. This phenominon is more often seen with the MBP tag as I have a similar vector with GST tag and I usually don’t see GST tag remain binding to protein after cleavage.
Have anyone experienced the same thing-MBP binding to target protein? If so, is there any good trick to remove it?
Thank you!
Lyra
I will try adding some maltose next time to see if it helps. Any other suggestions will be deeply appreciated!!
If there is an interaction between the two proteins, you may be able to disrupt it by increasing (or decreasing) the ionic strength of the buffer (i.e. the salt concentration) or changing the pH.
A good way to start would be to run the cleaved protein through ion exchange chromatography and elute with a salt gradient.
So your first step is to bind the intact protein to a Ni-column and elute with, what -imidazole?? If that is not removed (by GPC for example) it will prevent binding of his-tagged proteins after TEV digest. So that might explain your first problem.
I have no idea about the second problem.
I have dialyzed the imidazole out already & I usually don’t see same issue with GST tag.
Changing pH and ion strength is a good idea!
Thank you guys for giving me suggestions.
Hi Lyra,
1. The presence of unbound His-MBP in the flowthrough fraction may be due to the amount of the Ni-NTA resin is relatively less than that of your protein, or because the binding efficiency of the resin is not enough due to long-term use. If this is the case, it is recommended to increase the amount of resin or regenerate it.
2. There are cleaved protein (without His-MBP tag) in the Ni column bound fraction, which may be that your target protein non-specifically binds to the column due to incomplete folding, that is, although the MBP tag helps your target protein improve its solubility to some extent, the property of the target protein is still not stable enough. It is recommended that you try ion exchange to purify the target protein. If it is still not possible to separate the target protein from the MBP tag, other truncation or protein expression systems are recommended.
Hope it helps!
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