Hi Seyed,

However, your provided information about your protein is not sufficient, but still, I could suggest a couple of things worth trying. Could you please tell let a bit more about the origin of protein, expression system, protein tag, expression condition, temperature, lysis method and buffer composition? If you have already tried and optimized everything at the expression level (for example low-temperature expression, solubility tag like MBP, GST, SUMO etc, overexpression in presence of ligand, metal ions etc..), you can try a range of detergents to solubilize the protein from inclusion bodies. Urea is a strong denaturating agent and causes the unfolding of the protein. Using a high concentration of chaotropic results in complete disruption of protein structure, so better to replace UREA with some detergent like NP-40, Sarcosin, triton X-100, twin20, DDM etc. You could use a combination of these detergents to solubilize the protein and remove them step by step in further purification steps.

The extreme pH buffer has also been reported as a mild solubilization method. High pH (>12) buffer in combination with 2 M urea has been used successfully for solubilization of IB.

Use low urea concentrations with detergents like N-Lauroylsarcosine and Lauroyl-L-glutamate have also been reported to improve the yield.

If nothing works with your protein and you end up with UREA, then try to remove UREA step by step by following various refolding protocols. For example, Multistep dialysis against a large volume of refolding buffer in the presence of low concentrations of AAs like arginine, proline, glycine etc. Sucrose, sorbitol solutions.

Best of luck,

Have you tried using milder buffers containing Deoxycholate or sarcosine ( low concentration  0.5 % w/v) ? Although, harshly denaturing buffers with Urea and Guanidine hydrochloride work well to dissolve proteins in inclusion bodies, they can also cause the proteins to stick to each other and aggregate. These aggregates do not bind to the resin well. Another suggestion is that you an try to standardize the protein induction and expression conditions to allow for slower expression and better folding. This can minimize the amount of proteins stuck in inclusion bodies.

Hi Seyed,

1. You may try slow induction of the protein in lower temperature to have it in the soluble fraction.

2. The His-Tag of your protein is somehow not accessible for affinity chromatography. You may try ion exchange chromatography based on the pI.

3. To minimize precipitation, step wise dialysis of the protein is recommended so that it effectively renatures. For example from 8M urea to 6M, 4M, 2M and no urea.

All the best.

You need to slowly remove the urea to make the protein refold into correct structure. I'm not sure how you remove the urea, but you could try a few ways such as dialysis, chromatography, etc. More info on this: https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-lysis-and-extraction/handling-inclusion-bodies

https://lab.plygenind.com/how-to-solubilize-and-refold-inclusion-bodies-a-practical-guide