Hello Warren,

You can try tu use different buffers based on Tris-HCl, Hepes, PIPES, always using β-Mercaptoethanol or DTT. You can try sequential refolding from diferent dialysis bufers, in example, starting from 8M , to 4M, then to 2M urea and the final buffer, or using a buffer with 6M guanidinium chloride, then to 4M and/or 2M urea and then  the final buffer. However, sometimes refolding doesn't mean activity. Take care with those cysteines state. They are important for the activity. Moreover, ¿how many NaCl are you using? Depending on the protein, specially when the activity depends on the dimer formation, sometimes NaCl amount affects to the activity. You can try also Circular Dichroism  to check if the secondary structure is ok. You can also try to remove the His-tag (for example, using thrombine) to remove any possible interference.

Good luck

Hi Warren,

From what you have told I would say your IBs are not the 'classical' ones, otherwise you wouldn't be able to solubilize them in 2 M urea. Also, 1 mM DTT is normally not sufficient to get S-S bonds broken. Did you get these IBs at one of the low-temperature induction experiments? I guess you did.

For a 'classical' IB it is essential to start with complete denaturation which you can obtain with e.g. 7 M urea, 10 mM DTT, Tris/HCl pH 9. It is essential that you increase the pH above 8. Then you can try either the dyalisis or rapid dilution approach again.

Since you could solubilize your IBs at relatively mild condition, I assume you deal wih atypical ('non-classical') IBs for which a range of alternative protocols has been developed. Here, you assume your protein is just aggregated, not really misfolded. Consult the literature for details of protein recovery from non-classical IBs.

You will likely need a specialized E. coli expression system, see below link.

https://www.neb.com/applications/protein-expression-and-purification/expression-of-difficult-proteins/disulfide-bonded-protein-expression

Thank you all for enlightening me with your exceptional contributions. Let me apply and i will update you the outcome

For doing CD, u have to dialysis all the refolding buffer constituents, as CD is highly sensitive. Alternatively u can also confirm protein refolding by Fluorescence spectroscopy even in the refolding buffer. U can also try limited proteolysis, but again the dialysis had to be done after refolding. 

There are kits commercially available to refold your protein in several conditions at the same time. I have used the Thermo one and it Works.

Perform electrophoresis (Laemmli) without 2-mercaptoethanol or DTT protein samples before and after refolding. If passed кenaturation of the picture on the site of protein electrophoresis protein band appears. otherwise most of your protein is delayed at the start. As in sample inclusion bodies solution before addition DTT. For sample protein preparation add to protein solution 1/10 volume 1-0,5 M iodacetamide.

@Romanov Vladimir

when i perform reduced and non reduced SDS-PAGE before refolding, the band for non-reduced is relatively smaller than the reduced SDS-PAGE but on the correct position. however, for refolded samples, the non-reduced band is very minute and sometimes slightly below the expected size.

moreover, i always add DTT to solubilizing buffer because without it my protein doesn't solubilize even in strong denaturants like 8M urea and 6M GdnHCl. so i think running without 2-ME before refolding is not so conclusive in that DTT will still be there.

when i try to dialyse in solubilizing buffer (2M urea only) to remove DTT and perform the DPPH antioxidant assay, i could see some activity but when i decide to dialyse in PBS, no activity even when i include GSH/GSSG in 1/10 ratio.

Everything is fine. You have a small part of the protein denatures. Try the renaturation and dialysis at 4-8 degrees Celsius. Time renaturation 4, 8, 16, 24 hours. Collect samples at a specified time and add iodoacetamide. Iodoacetamide blocking SH  group of of 2-mercaptoethanol, and the remaining SH group of SH protein. Stops refolding in Your samples. Spend electrophoresis and see results. Concentration GSH/GSSG should be 1 / 0.1 or 10/1 mM. Lower the protein concentration to less than 0.1 mg / ml.

Another way of annealing. After dissolving the inclusion body in urea or guanidinium chloride, and reducing DTT  or 2-mercaptoethanol solution, dilute 10 times with cold 50 mM Tris-HСL, pH 8. The final concentration of protein is less than 0.1 mg / ml. Further samples were taken, as I cited above.

Thank you and i will come back with the update

Hi Warren,

It looks like you are approaching this in a correct way. I agree with others that you should denature in 8M urea or 7 M GuHCl to avoid having partially misfolded proteins going into the refold buffer.

One thing you haven't mentioned is what happens after the removal of denaturant. Do you get a cloudy solution or does your protein aggregate into a 'scrambled egg' state? 

In either case addition of a detergent such as Brij or Tween-20 at just above CMC might help. Sometimes a small quantity of SDS can also help. We always find TCEP better than other reducing agents at refolding. Also ensure your final pH is at least 2 points above the pI and use the right buffer for the pH e.g. Tris and PBS are not good at high pH and it is better to use ethanolamine-HCl (or similar) for pH 8.5 and above.  

When you spin out any aggregates do you have a good proportion of soluble protein? If so the lack of bioactivity implies misfolding or lack of co-factors - you don't say what the bioactivity is and how you assay for it but sometimes proteins extracted from native tissues already co-purify with any co-factors etc that are necessary for activity. In the case of proteins from heterologous expression systems, especially if denatured and refolded, they may need extraneous addition of such co-factors whether they are metal ions, small proteins etc. 

The other possibility is that something in the protein refolding buffer is interfering with the bioactivity. Can you pass the soluble fractions through a PD10 column and buffer exchange to remove residual denaturant and other components such as arginine? Does this help the bioactivity?

I hope this is useful for you!

Deepan

Hi Deepan,

Thank you for your comment

 When i remove the denaturant i get a cloudy solution. but in refolding buffer aggregation appears in the first few hours and disappears after overnight.

By saying "TCEP better than other reducing agents at refolding," are you implying using TCEP instead of GSH in refolding buffer or instead of DTT in solubilizing buffer?

however, in instances where i get aggregate after refolding, a clear solution is obtained after a spin and i don't get a good proportion of soluble protein; Bradford assay or BCA gives negative values ( i guess that means no protein present in solution).

My protein is antioxidant, so bioactivity with DPPH antioxidant assay and once i have the activity then i will need to proceed with MTT assay.

Hi Warren,  

I meant TCEP in the refolding buffer.  

It looks like you have a zero refolding yield at present. Try some of the things suggested by your correspondents here and see if that improves. If there are any cofactors for your protein try adding them to the refolding buffer - sometimes proteins that are heteromeric need their partners to refold correctly. 

I don't know anything about antioxidant proteins in particular but if you check whether your protein or other structurally similar antioxidant proteins have been refolded previously by searching the literature, you may find the answers.