I have a GST-fusion protein (95 kDa) that forms inclusion body in Ecoli BL21 expression system.
I couldn't solve inclusion body formation with reducing temperature and IPTG concentration, hens I use of different methods for the solubilization and refolding the GST-protein.
1. 6-8 M urea and dialysis ( in this method the yield of protein was very low and protein was very unstable)
2. 2% Sarkosyl for solubilization and sarkosyl:Triton (1:20) for binding to Bead for purification (in this method the yield of binding to bead was very low)
3. 10% sarkozyl for solubilization and sarkosyl;Triton;Chaps for binding to bead (solubilization is well but yield of active protein is not efficient, too)
Do I have another way for this?Does anyone help me for solving this problem?
I discourage you to continue thinking about refolding...Either you switch to an Eukaryotic expression system (i.e Saccharomyces or pichia) or if the problem is disulfide formation in E. coli then read further below.
The use of GST-fusion strategy may not help if the protein need special disulfide bridge formation (i.e. "trans" bounds). Furthermore for "cys" disulfide formation they could happens easyly in the cytosol of special E. coli strains bearing "gor" mutation.
I recommend for your purpose, SHuffle® Competent E. coli from NEB but these still are not completely protease free. If you have problem with proteolysis try Origami B(DE3) cells (Novagen) or Rosetta-gami B(DE3).
If after these try you end up intoyour protein still present in the inclusion bodies only, try the pichia system.
Good luck !
I discourage you to continue thinking about refolding...Either you switch to an Eukaryotic expression system (i.e Saccharomyces or pichia) or if the problem is disulfide formation in E. coli then read further below.
The use of GST-fusion strategy may not help if the protein need special disulfide bridge formation (i.e. "trans" bounds). Furthermore for "cys" disulfide formation they could happens easyly in the cytosol of special E. coli strains bearing "gor" mutation.
I recommend for your purpose, SHuffle® Competent E. coli from NEB but these still are not completely protease free. If you have problem with proteolysis try Origami B(DE3) cells (Novagen) or Rosetta-gami B(DE3).
If after these try you end up intoyour protein still present in the inclusion bodies only, try the pichia system.
Good luck !
Before refolding, I suggest you to ensure that there is no GST fusion tag cleavage upon denaturation. The GST tag may degrade and loss its ability to bind to the IMAC resin under high urea concentration. If this happens, You may optimise the urea concentration used during solubilisation and purification. For in vitro refolding, you can visit this website: http://refold.med.monash.edu.au/, this will give you some hints on selecting the best refolding protocol which is most related to your protein. In my experience, dialysis refolding works for me using decreasing urea concentration (usually takes more than 1 day). You may include glycerol or other additive (Triton X and BMe) in the refolding buffers in order to prevent protein aggregation. Goodluck for your research!:)
The REFOLD database as Kelvin suggests is useful, though its data sets are a bit old. There are many publications regarding refolding and solubilization strategies which you can find them easily on google scholar (e.g. http://www.ncbi.nlm.nih.gov/pubmed/16233795). I would point out some notes, so I wish you find them helpful:
For solubilization:
1- Regarding sarkosyl you should remember that you cannot solubilize more than about an equal weight of protein per weight of Sarkosyl. For example, if you have a large washed IB that contains 150 mg of target protein, it will not be solubilized completely by 20 ml of 0.3% Sarkosyl (60 mg of Sarkosyl). Also remember that SDS is highly poisonous, so in later steps of purification you would run into many deep problems.
2- Many studies recommend use of 8 M urea or 6 M GdnHCl as solubilization buffer. A general solubilization buffer for IB's includes 6 M GdnHCl, 100 mM DTT (to break disulfide bridges), 1 mM EDTA and 50 mM Tris-HCl.
For refolding:
1- There are many refolding strategies including on-column, dialysis and dilution. Among which the dilution is the most common and straight-forward method. However please note that on-column methods show better yield compared to other methods, and I recommend to forget about dialysis which its performance is low.
2- A general refolding strategy is to dilute your solubilized IB's (at low concentrations of 100-1000 ug/ml) into refolding buffer to achieve a ~50-100 fold dilution. A refolding buffer may contain low concentrations of urea (1-2 M), refolding additives (e.g. L-arginine ~0.3-1 M), redox agent (e.g. GSSG/GSH) in case of proteins with disulfide bonds, and 1 mM EDTA and 0.1 M Tris-HCl.
Please note that solubilizaion and specially refolding strategy are protein specific and for each target protein the buffer compositions must be optimized separately. Therefore, you need to optimize the suitable refolding buffer for your protein. I recommend to assay your protein after each experiment to ensure its activity (the same goes for solubilization yields using various techniques such as CD, Western Blot, etc.).
Good Luck.
If your protein is extracellular and forms disulphide bonds I recommend using an oxido-shuffling system.
I had an extracellular protein that formed tough inclusion bodies in E coli, and so rather than purify with the affinity tag I just purified the inclusion bodies, then dissolved and refolded them directly. Details are in this paper:
https://www.researchgate.net/publication/257770359_1H_13C_and_15N_resonance_assignments_for_the_fibrillin-1_EGF2-EGF3-hybrid1-cbEGF1_four-domain_fragment?ev=prf_pub
Article 1H, 13C and 15N resonance assignments for the fibrillin-1 EG...
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