[Papaver]

Hi everyone,

I would like to ask you for some ideas or advice. I have huge problem with removing a tag from a protein. Here’re the facts:

It’s a MBP-tag that should be removed. PreScission protease is used (I can’t use factor Xa because the size of Xa chains and protein of interest are too similar). Reaction was performed overnight at 4 °C

Issues:
1) Only half of MBP-Fusionprotein is cut by PreScission.
2) Ignoring the incomplete proteolysis I performed the reaction onto the amylose resin column. In theory the cut protein has to be found in the flow-through whereas MBP and remaining MBP-Fusionprotein are still bound to the amylose resin. BUT: Untagged protein is eluted together with MBP and Fusionprotein.

Things I tried so far:
Adding to proteolysis: 1.5 - 2 M Urea or 500 mM NaCl or 0.1 - 1 % Triton X-100 or 0.1 - 1 % Tween-20. Using PreScission buffer althougt it's not ideal for my protein. These experiments were all done in Eppi-reaction as well as on-column reaction.

Further: reduced concentration of protease inhibitor, extended reaction time, added fresh protease after overnight incubation, increased protease concentration, introduced a linker sequence (ser gly gly) between MBP and PreScission site and between PreScission site and protein.

Gel filtration und normal conditions seems to be useless because the cut protein sticks to the MBP-fusionprotein as if protein domains are very much interaction with each other.

If anybody has further ideas how I can complete the proteolysis or how I can weaken the protein domain interactions please let me know.
Unfortunately the protein seems to be only solube as MBP fusion thus changing the tag is something I will try only to be sure but it’s probably not the solution.

[Yggdrasil]

During the proteolysis step, you should basically have no protease inhibitor present.  After binding your protein to the MBP resin, you should wash with multiple column volumes of a buffer containing no protease inhibitor at all before adding your protease.

[Papaver]

I washed the column after binding to the resin with 10 column volumes and therefore used buffer without any protease inhibitors.
The result was the same (PreScission activity is not inhibited by protease inhibitors to a certain concentration and I calculated the concentration to make sure it's not too much)