I have a recombinant protein, which I purify using Ni-NTA chromatography in denaturing conditions (8M Urea). It comes in high concentrations, but upon refolding there is aggregation and loss and the concentration drops to 0.1 mg/ml. Any suggestions as to how I can prevent this from happening.Thanks in advance
This depends on the process you are following for refolding. If you remove the denaturant (8 M urea) by stepwise dialysis (decreasing the urea conc stepwise in the dialysis buffer) then it increases the yield of the protein.
I have tried step wise dialysis, as well as rapid dilution.
then concentrated the protein using amicon ults concentrators.
by then the concentration goes down rapidly,
Try refolding the protein while it is still bound to the Ni-NTA resin by gradually decreasing the amount of urea from 8M down to 0 M urea. Then elute the protein with increasing amounts of imidazole. This has work well for several proteins we work with.
using on column refolding was not suitable to my protein as i lost all the protein with nothing being there during elution.
is there any other way?
i tried even using 10% glycerol during the dialysis but still the aggregation was ineveitable.
A common approach to refolding is to refold the protein first into a solution that contains a "refolding agent", which helps to refold the protein. Common refolding agents are 0.5 M arginine pH 8.5, and sarcosine. You can also buy kits of refolding conditions to try in small scale, if you have a good method of detecting activity (Novagen and Pierce both sell these).
The usual way of operating with these is either to slowly add the unfolded protein, dropwise, to a 10-fold excess of the refolding agent; or to dialyse against the refolding agent. The refolding agents are usually pretty adept at keeping misfolded protein in solution, so again you then have to dialyse against buffer. Unfortunately, most of your protein will drop out of solution here, but hopefully you still keep enough to work with.
If your protein contains disulfides, you should also include a redox buffer (e.g. 5 mM reduced glutathione, 0.5 mM oxidised glutathione), and reduce your unfolded protein (1 mM DTT), as otherwise the cysteines are unlikely to re-form properly.
To be honest, if you are keeping protein to 0.1 mg/mL, you are probably well on the way to a good solution - you at least have something to work with! That is treating the protein fairly harshly, so if you are a little more gentle, you may well find that this is enough to give you all the protein you need.
If your protein has a cofactor that it uses for folding include it in the buffer. Heme B for instance helps the folding of some hemoproteins giving them more structure.
i purified the protein again using 6M urea, and for refolding put it for dialysis.
post dialysis the protein got aggregated, i centrifuged at high speed and separated the aggregate from the solution, the aggregate in the tube , i again subjected to denaturing conditions and refolded again by dialysis, but this time i added 5mM arginine to the dialysis buffer.
i checked the protein on SDS page gel and estimated it using Lowry . I got a single band and the concentration was around .4mg/ml, and i have around 4 ml of this solution.
I wanted to know, what concentration of Arginine is ok to be used and what other ways i could use or stabilizers to prevent aggregation,
i need this protein for invivo and invitro assays so the buffer i maintain it in is PBs.
thanks in advance for all the suggestions :)
This after dialysis of the aggregated protein and refolding with arginine.
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