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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
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.
Hola, unless it seems silly here and I have seen some references about it in some forum, it´s better make digestion outside colum and after recharge the column with digestion and you will have your protein in the flowthrought, be carefull in pass the sample at low flow and be sure of don´t saturate the column. collect fractions and analize, if you have contamination gel filtration is an alternative but it has sample volume limitations. an ion exchanger could be better. Buena suerte
Thanks for your reply.
If I do the digestion outside I cannot reload the fraction onto the column because of the maltose in the purified fractions. And I cannot perform the digestion with the crude extract because of the mass of other proteins. So for me it's the best way to make it onto the amylose resin column. And it would work out but the cut part of the protein remained stuck to the uncut fusion protein very strongly.
Ion exchange would not work either because the pI of MBP-protein and protein is the same.
Gel filtration test run as a gravity flow experiment indicated that all components are in the same fractions.
Is there any possibility to perform a gel filtration under changed / drastic conditions to resolve the interaction?
Are the 2 proteins very different in size?
If so you can try to do your cutting out of the column and add whatever to the solution to resolve interactions (8M urea, 6M Guanidium HCl, your choice of salts and detergent?) and then dialyse the hell out of if your protein is smaller than the MBP it or use a selection of Amicon filters (or something similar) in cascade to 1st get your protein in the eluate, and then wash it up in a filter that would keep the protein the saved fraction.
Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
I would use urea or guanidium as a very last option, if he's going for active protein. Better is to use high salt to brake the electrostatic bonds.
Papaver, you can always dialyze the maltose away, so that you can load it repeatedly.
How do you know that it was partially cut, if you're not able to separate the cut and uncut proteins?
Cis or trans? That's what matters.
I've already tried everything: urea, guanidium, sodium deoxycholate, triton, tween, even acetonitrile (to separate the proteins). I also tried high salt (up to 700 mM NaCl), low and high pH (4.5/9).
I know that it's partially uncut because I got three bands after cutting: one of MBP-Protein, one of MBP and one of protein. I also did western blot and there I got two bands (MBP-protein and protein). The bands had the expected sizes.
And, I already know that my protein highly aggregates. DLS measurement showed huge but stable aggregates which surprised us. This would explain why it is so hard to cut it completely or why so difficult to perform gel filtration (always found in void volume) and run native gels.
did you try both termini of your protein? Maybe it would be easier to produce your protein with no tag and just purify through several chromatographic steps. It just may be that the terminus of your protein should be somewhere inside of the structure and thus it cannot be efficiently separated. (however, then the denaturing agents should work).
Cis or trans? That's what matters.
It might be worth trying to denature your protein then run the digestion with a Western to confirm that the protein being made in stable active form. The cells that you are using to express the protein may be having a trouble making the protein in the form that it would normally be found and so are folding it incorrectly. The tags as JackBean said may be internalized or causing folding to be inhibited in some way. I would make sure that they are making the protein of the right MW.
I have seen the same problem in GST and GFP and it's incredibly frustrating. Best of luck!
The untagged protein is mostly found in inclusion bodies so it would be difficult to purify it. Anyway it might be worth trying it.
Luckily I can express the protein in its native surrounding, of course the amount of protein (especially in the tagged form) is unnatural.
Anyways, thanks for all replies. I will see what happens next
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