I have quite an annoying problem - I'm purifying a protein with a His-tag with elution buffer (6M urea, DTT, 300mM imidazole). In elution buffer, the protein is in the supernatant and everything seems ok but when I'm starting dialysis against 2M NaCl, DTT to refold protein it starts precipitating. I change the buffer very often (every 2h) and mix samples in the dialysis bag but still the protein precipitates. Do you have any idea what could be wrong with it? If any more information is needed just let me know. Thanks.
I'd slowly decrease the urea while slowly increasing the NaCl - keep the DTT concentration and the pH constant throughout.
The protein remains soluble in your elution buffer because it contains 6M urea.
Upon dialysis incorrectly folded protein typically precipitates, whilst correctly folded protein will remain soluble. You then require a assay to test the refolded protein. Concentrate on what remains soluble. Refolding is tricky at the best of times. Usually I try refolding by either dialysis or rapid dilution with a final protein concentration of 1mg/ml or 0.1mg/ml. Look up protein refolding kits if you get completely stuck. I've used them they work well.
Urea is chaotropic agence and when you remove it the protein is not soluble any more that could hapened, you can try to use lover urea like 4M and you can use sach protein in many reactions. Or you can try to use several detergent to support the solubility, depend on what you plan to do later
You could be at or close to the proteins isoelectric point. That could be determined with various analytic techniques. Try going above or below the isoelectric point to start, where the protein will be more soluble and then move slowly toward a pH where you think the protein could be folded correctly. Do this in as dilute a situation as possible using your dilution and reducing agents to prevent inappropriate crosslinking.
Check if you have cysteines in your construct that are supposed to form disulfide bonds. With all the DTT present in your assay disulfide bonds can't form properly. Reduce your inclusion body protein in 10mM DTT but then lower DTT conc to 1mM when you purify on column. Then add a redox system such as reduced and oxidized glutathione to refoding buffer and try refolding o/n by rapid dilution (to form SS bonds). Leave the DTT out at this stage. Then dialyse the refolding mix to get rid of Urea. Also step-wise dialysis to lower Urea conc might help. But check on the disulfide issue first.
I agree with Joseph: always do a quick check on the isoelectric point of the protein to make sure the buffer's pH is far enough away to avoid precipitation (misfolding or other issues notwithstanding). I like this tool: http://web.expasy.org/compute_pi/
Check pH of buffer: isoelectric point can be an issue. Again casual refolding of concentrated cystenie-rich proteinsis with Cys-Cys misparing is always a problem!
No other suggestion... you can try to dilute proteins before dialysys, if feasible... but solution seems not easy.
Can you avoid urea denaturation?
I'd try this approach: it's better to avoid dnaturation if re-naturation is difficult...
some proteins precipitate in high salt concentration. You could perhaps try to lower your NaCl concentration. Good luck!!!!
Thanks for your replies. So: protein pI is 10, buffer has pH 7,5. People who worked on this protein before told me that refolding of it is really easy and they are suprised that it precipitates so easly. DTT is added to prevent oxidation (no sulfate bounds in protein). 2M NaCl should be fine (almost 30years people expressed and puryfied it that way). I will keep looking for my mistake and thank you for many interesting answers
I agree with Mr. Mihir and Mr. Ologbenla. Lowering the NaCl concentration may help. You can also check for the presence of ions (like Ca or K) in the water you are using. A few months ago our lab also faced a similar problem while purifying a lectin.
I agree with the above concerns about , conc. of Buffer (eluting,Dialysis) DTT, Cysteine Disulfide etc.Are you Dialysing at room temperature or below, Maybe if Temp. is not an issue you could try dialysing at 35-40 C . Worth a try only if it is not a Protease otherwise it will self degrade.
Adding some EDTA (1-5 mM) to your dialysis buffer wouldn't hurt. If you have heavy mrtals (like Fe, Zn, Pb,,,) contaminating the NaCl, these could stick yo the His tag and promote misfolding/aggregation
I suppose I know what was the problem - too high protein concentration. In supernatant I had properly folded protein and pelet was protein which cannot be properly folded. Finally I have properly folded protein in 1M NaCl and everything seems ok. Thank you once again for your replies.
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