Happy New Year to all of you!
I was wondering if anyone would have some suggestions for my problem.
I am trying to purify a GST-tagged protein but the protein is strongly bound to the glutathione beads. While I am able to release a small amount of protein with reduced glutathione, most of it is still attached to the beads. I digested the GST-tagged protein to separate the protein from the GST but the problem is the same as my protein is still strongly attached to the beads.
The protein is expressed in bacteria and is partially soluble; it is intended to be used in ELISA as a tagged or untagged protein.
Any suggestion would be much appreciated!
Thank you very much.
we had this problem several times, upscale was not possible. It was obvious that the protein was not properly folded.
Have you adressed this by SEC or other methods?
Many thanks for your reply. You are right, it is very likely that the protein doesn't fold properly but I haven't confirm it yet.
The researcher who asked for the protein would like me to give him the protein the way it is now so he can test it. It is indeed the first time that there is a large amount of this protein expressed that can be purified in a single step. A previous His-tagged version of the protein was very very poorly expressed.
To give some protein to the reseacher I need to find a way to detach the protein and I am planning to do some tests next week starting with mild detergents. I am going to try other chromatography methods as well.
At the same time, I am thinking of trying a different tag, would you suggest one or do you think I should try directly to express the protein in a different system (mammalian cells)?
Thanks again for your reply.
You could try using a His-GST tag as the GST worked to increase your expression and then use the His for purification instead of going to a whole new tag. GST can be a problem with incorrect folding of your protein of course.
For us, we quite like the SUMO tag and find it doesn't give many false positives ie folded tag and unfolded protein.
Thank you for your reply. I am planning of testing if the GST-tagged protein could binds to Nickel beads in a non specific way before using a tandem His tag as you suggested. I am also thinking about trying SUMO or NusA.
Many thanks for your suggestions.
I doubt it is worth the effort to release a protein that is aggrgated on the beads. I would instead invest time to express properly folded protein either with other tags like Sumo, MBP , NusA eg or other constructs or other hosts.
I completely aggree with you. I am preparing my primers right now to put the GOI in your pCoofy plasmids starting with Sumo and NusA.
Thanks again for your help.