I am trying to purify and refold an insoluble protein from E. coli cells. I solubilized and purified the protein using 8M Urea. After that, I pooled the eluted fractions containing proteins to sequential dialysis in 6M, 4M, 2M, and 0M Urea at room temperature. I need the protein to perform structural characterization, for which properly refolded protein is required. However, when I concentrated the dialyzed protein using Centricon and performed CD Spectroscopy, the spectrum was similar to that of the buffer only. But on SDS PAGE, I could see a distinct protein band.
After refolding, dialyzing, and concentrating the protein, it would be a good idea to run it through a gel filtration column to separate soluble, aggregated protein (which elutes in the void volume, typically) from monomeric protein eluting later.
Try to dialyze your protein not in centricons, but in classic membranes bags or slider. Use the slow moving from 8M Urea (or 7 M Guanidine) to 0 M through steps: 8.0 - 3.5 - 1.2 - 1.0 - 0.0 M (every steps should take at least 2 hrs. The transition 3.5 -> 1.2 M could be done even over night). Moreover, just to use two basic buffers which you will mixed up to reach right proportions: from START-buffer [8 M Urea (or 7 M Guanidine), 1x PBS, 0.2 M L-arginine, 5% glycerol] to the END-buffer [1x PBS, 0.2 M L-arginine, 5% glycerol] with pH = as you need. And last step - just change END-buffer to the fresh one or other that you might prefer to remove L-arginine or glycerol or whatever to add.
Aakriti Singh try to solubilize inclusion bodies with your protein in such buffer: [7 М Guanidine-HCl, 0.25 M NaCl, 0.037 M Na2HPO4, 0.1% b-ME] pH = as you need, then dialyze it as I describe above
Yuri Cheburkin I usually perform sequential dialysis using dialysis membranes (3kDa cut-off) i.e. 8.0M - 6M - 4M - 2M - 0M at 2h duration at room temperature. However during the last step, 2M - 0M, my protein starts aggregating, with visible milky solution appearance. I have not used L-Arginine as of now, and I am curious to know if adding that during the last dialysis step would be helpful.
Aakriti Singh the L-arginine(-HCl) is a kind of aggregation suppressor. It is better to keep it in refolding buffer during whole time of refolding process. The refolding itself is something that needs a time, do it too fast is probable wrong. Please, check this two publications, they might help you in your deal - doi: 10.3390/biom4010235 and 10.1016/S1046-5928(02)00641-1
Aakriti Singh BTW, if you get milky solution at the end of refolding - just separate it and check where is you protein is. It could be in pellet, in super and in both forms, then take a decision. If in pellet, just resolve it in GuaHCl and dilyze again, but add some glycerol in buffers of final steps (2 - 5%). This could be successful
Aakriti Singh you can use either L-Arg or L-Arg*HCl. Just look after pH of buffer
Dear Aakriti Singh
Correct me if I’m wrong but you want to study a protein that is intrinsically insoluble in water/buffer (or at least with a low solubility)? Why not keep it in the 8M urea and dilute it (out of your protein stock solution) in whatever assay you want to study.
See for example how a membrane protein was studied this way (and the possible effect of a chaperone): Article In-vitro studies on the folding characteristics of the Esche...
Or how the same protein is studied in a variety of other (biophysical) approaches:Article Mode of Insertion of the Signal Sequence of a Bacterial Prec...
Just an idea which might help.Best regards.
PS. Did you check what prediction programs might tell you, in terms of helical content etc. see for suggestions for example my reply here on RG https://www.researchgate.net/post/Why_do_I_get_different_result_for_the_prediction_of_protein_secondary_structure
Or to check what kind of a protein you have (water soluble, amphipathic/surface seeking, membrane protein): Article New User-Friendly Approach to Obtain an Eisenberg Plot and I...