Removing MBP tag
Posted 10 August 2011 - 05:53 AM
I would like to ask you for some ideas or advice. I have huge problem with removing a tag from a protein. Hereíre the facts:
Itís a MBP-tag that should be removed. PreScission protease is used (I canít use factor Xa because the size of Xa chains and protein of interest are too similar). Reaction was performed overnight at 4 įC
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 itís probably not the solution.
Posted 17 August 2011 - 08:00 AM
Your description matches a phenotype of insoluble/not correctly folded protein that is kept by MBP in solution due to the tag's passanger solubility effect.
Does the size of the fusion protein on size exclusion column correspond to the theoretical one? I had a case where the fusion appeared as a soluble aggregate where even though it was staying in solution it was going in the void volume of appropriate size exclusion column. Another way to verify the size is to simply do dynamic light scattering measurement of your purified fusion protein.
You could try to run an ion exchange column. The advantage here is that you will most likely separate your target protein from MBP even if they are similar in size.
Edited by prodes, 17 August 2011 - 08:06 AM.
Posted 23 August 2011 - 04:19 AM
So far the fusion protein was always found in the void volume of the size exclusion column. This is one reason why I would like to remove the tag. Ion exchange column would probably not work since the pI of all components were determined to be equal.
Dynamic light scattering measurement may be a good possibility...
Posted 23 August 2011 - 06:06 AM
In your situation I would still try with IEX since it may separate even proteins with theoretical pI but different conformations.
What studies do you need the protein for and what type of protein it is? If it is an enzyme and you need to do some kinetics you most likely are fine without removing the MBP. Moreover, if this protein has any kinda of activity/binding this might be another way to check if it is correctly folded in the fusion.
Finally, if all these rescue strategies are unproductive you may reconsider how you express it (other tags, strains, organisms, refolding, etc.)
Posted 24 August 2011 - 12:16 AM
I need the protein for structural studies, 3D as well as 4D structure. The protein is a negative regulator und inhibits the transcription initiator of nitrogen fixation. Additionally it is able to interact with the membrane, thus it's a very hydrophobic protein.
I think the protein is folded properly because I have already done in vitro experiments with purified protein and I haven't changed anything in purification protocol. Former efforts to purify the protein using other tags failed due to unsolubility but I guess since there exist new possibilities I will repeat trying another tag.
It might be right that I should at least try the IEX exspecially since I've already tried so many things.
Posted 23 February 2012 - 11:23 AM
Unfortunately I am facing the same problem with my protein as the one mentioned by you. have you found any solution to it?