i try to produce a protein fragment which contains numerous cystein aminoacids and two beta-sheet structure. This fragment was produced in BL21 strain and is retrieved in the soluble fraction. But this fragment seems to be not properly folded since it have lost their binding activity...how can i try to refolding this protein ? with a protein refolding kit ? thank you for your help
Hi there,
The first step will be to denature your protein completely to eliminate any present misfolding in your sample. Then different techniques are available (dilution, dialysis, on-column refolding...) to remove the chaotropic agent and to introduce a GSSG/GSH system in order to allow disulfide formation if needed.
With numerous Cys residues, the opportunity for formation of incorrect disulfide bonds is high if the protein is allowed to oxidize. If the protein is normally cytoplasmic, and therefore in a reduced state, there should not be any disulfide bonds. Therefore, the protein should be kept reduced at all times. If the protein is normally not cytoplasmic, it probably has disulfide bonds. In order to make sure that the bonds are formed correctly when the protein is refolded and re-oxidized under controlled conditions, you need to find out what the correct disulfide bonds are in the native protein.
Hi Nicolas, if this protein is eukaryotic, you should maybe look into expressing this protein in insect cells. Those cells have chaperones and disulfide isomerases.
First work on your protein to get in the insoluble fraction. Expression at high temp usually misfolds the protein and it will be in pellet fraction.
Then, try protein refolding using standard protocol.
- Badges
- Science topic