I am working on a truncated viral protein. I have expressed my protein in pET15b system, most of the protein is insoluble thats is in the inclusion bodies. I used 8M urea to dissolve the pellet and then refold the protein on the column. pH of all refolding buffers is 8.5 and elution buffer is 250mM Imidazole pH 7.5, pI of my protein is 6.9. I had about ten elutes of 500 ul each, and I see aggregates in at least first four elutes. On coomassie stained gel, I can see about 3 mg/ml of protein in first few elutes. I did dialysis on protein after spinning the aggregates down and I saw some precipitates. How can I standardize the elution conditions to keep my protein soluble?
Reduce the concentration of the elution buffer. Insoluble proteins as such readily precipitate therefore, greater concentration of ions is not desirable. Its best to try an alternate buffer if decreasing the concentration does not help!
A possible problem is with the refolding itself, rather than the elution. The protein may not refold well on the column, so that it aggregates when eluted. You may need to change the procedure, using a different refolding buffer or changing the timing or changing the temperature. It might help to bind the protein to the column at a lower density by mixing a larger amount of the resin into the protein solution, then pouring the column.
You may want to try refolding in solution instead, by diluting the urea-denatured protein dropwise into a large volume of refolding buffer with rapid mixing. Dialysis can be used in order to slowly reduce the urea concentration.
There are also kits that allow you to test various reagents to assist in refolding.
I'd complete the purification under denaturing conditions and screen for refolding solubility in a DOE. A 1:20 dilution into refolding buffers and screen for movement of the protein through a small MWCO spin filter has proven a good method, larger aggregates will be retained or precipitate into the membrane and 24 conditions can be screened in a couple hours in a typical microfuge with A280nm monitoring. Keep in mind that TCEP will be needed during the purification if there are aberrant disulfides and then a GSH/GSSG mix in the refolding.
There are no "standard refolding conditions" you must explore a rather complex experimental space to get refolded/soluble protein,. Refolded/soluble does NOT necessarily mean it has a native fold either.
Thanks Everyone for your valuable suggestions.
Adam: I will try to refold the protein in-solution using slide dialyzer and see if it gets any better.
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