I am working on the expression of one protein with a MW of 22 KDA. This protein has 8 cysteines, pI: 8.58 and positively charged. This protein forms incusion bodies, I have done washes of the bacteria pellet with 50mM Tris-HCl, 100mM NaCl, 5 mM EDTA and then resuspended the cell lysate in 50mM Tris-HCl, 100mM NaCl, 5 mM EDTA, 8M Urea.
I have done aliquots (1 ml) of this supernatant and I tried to refold the protein. I added drop wisely one drop (10 microlites every 30 s) in a 50 ml refolding buffer (50mM Tris-HCl,, 50 mMArg, 50 mM Glu, 5mM/0.5mM red/ox glutathione) with gently stirring and let the solution overnight. I centrifuged and there is a gel-like pellet.
I have done SDS-PAGE analysis and the aliquots with 8 M urea contains the protein, as well as the gel-like pellet. But not the supernatant after the refolding step and it is not solubilizing.
Any good protocol to refold the protein or any suggestions to get the expected result? We have no idea if all the cysteines of the protein form disulfide bonds.
Do I need to add DTT in any of the washes or at the moment of add Urea?
All the best
Refolding proteins is really an exercise in getting lucky. If your protein is natively cytosolic, then I would include DTT in the denaturing and refolding solutions. If it is an extracellular protein or located in another oxidative environment, then it might be worth experimenting with different GSH/GSSH ratios...otherwise I wouldn't bother with that. Some suggestions for refolding are listed below:
1. Try using guanidine-HCl instead of urea. Urea can covalently modify lysine residues
2. If you can, try binding your protein to a column and refolding the protein while it's on the column by rapidly changing to a solution that doesn't contain denaturant
3. Instead of a rapid dilution of the denatured protein, sometimes a slower approach can give better results...try dialysis
4. Continue with your dilution approach, but use different buffers. Test different pH values and ionic strengths. Also, you can try using intermediate concentrations of denaturant (ex. dilute into a solution of 0.5M guanidine HCl).
Unfortunately, there's no guaranteed method. I've beat my head against a wall for several refolding projects and have had a low success rate myself. If I were you, in addition to continuing your refolding trials, I'd work on trying to express my protein solubly. Ideas are below:
1. Express at lower temperatures
2. Try a different expression cell line
3. Clone your protein under a less powerful promoter
4. Clone a homologous protein from another organism
5. Some combination of 1-4
Hi Jose, David and James, I started a thread on RG that I hope would bring together people having problems with protein purification from inclusion bodies.
https://www.researchgate.net/post/Why_are_so_many_researchers_trying_to_isolate_proteins_from_the_tangled_mess_of_coli_inclusion_bodies
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