How to renaturate my protein after urea treatment?

You can use this helpful protocol for refolding your target protein

Protein Refolding

Prepare 250 mL of buffer E (3 M urea, 100 mM Tris-HCl pH 8.0, 0.4 M L-arginine monohydrochloride, 20 mM reduced L-glutathione, 2 mM oxidized L-glutathione) in a 500 mL beaker and cool the buffer down to 4 °C.

Add 60 mg of denatured protein mix containing His6-Tlp3-LBD obtained in step 2.13 to 250 mL of buffer E, while stirring the buffer at 500 rpm. Incubate the refolding mix at 4 °C with continuous stirring at 500 rpm for 24 - 48 h. The final protein concentration in this step is 0.2 mg mL-1.

Prepare 7 L of buffer A in an 8 - 10 L bucket and cool it down to 4 °C in preparation for the next step (step 3.4) that will take place in 24 - 48 h (see the flowchart in Figure 1).

Immerse an ~50 cm piece of dialysis tubing of 28 mm inflated diameter, with molecular weight cut-off at 12 - 14 kDa into 200 mL of 10 mM ethylenediaminetetraacetic acid disodium salt (EDTA-Na2) and heat it over a gas flame until boiling. Rinse the dialysis membrane with 200 mL of ddH2O.

Clamp one end of the dialysis membrane with a dialysis tubing closure and transfer the 250 mL refolding mixture obtained in step 3.2 into the dialysis tube. Close the open end with another clamp. Check the integrity of the membrane to ensure there are no leaks.

Place the dialysis tube in a dialysis bucket with the pre-cooled 7 L of buffer A prepared in step 3.3. Place a magnetic stir bar, ensuring that it will not touch the dialysis tubing while stirring.

Dialyse the sample at 4 °C with continuing stirring at 500 rpm and change the buffer at least four times over a period of 12 h. After the last buffer change, leave the sample to dialyse overnight. NOTE: During dialysis, the excess of urea (~3 M) is gradually removed from the denatured protein solution, which allows the protein to refold. Heavy protein precipitation can be observed at the end of dialysis.

Remove the dialysis tube from the bucket and transfer its contents into a 500 mL beaker. Keep the protein solution on ice, unless indicated otherwise.

Filter the protein solution through a 0.43 µm pore size membrane into a 500 mL glass bottle to remove any precipitated protein.

For more details please refer to this open access article

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755555/

Thomas Svoboda

You could try to directly refold the protein on the column using a three buffer system (Urea-buffer, Tris-buffer, elution-buffer; the pH you need for the buffers depend on your protein of interest). Load the protein on your column under denturating conditions (UREA-buffer). Once the protein is on the column exchange the UREA buffer to one which is suitable for your protein (Tris-buffer) over 20 column volumes (refolding). Then wash the column over 10 more volumes and elute in a gradient with your elution buffer.

Good luck!

Asadollah Asadi

Equlibrium dialysis with apprprate cutoff and buffer under cold temperature is helpful. For increment refolding yield, also artificial chaperon and desiging protein specficic refolding buffer and condition is useful.

Yours truly

Manuele Martinelli

Dear Jihene Maati

Sincerelly, in generall i'm doing all the possible trials to produce the protein in soluble fraction and avoid refolding, if possible.

However if this is not possible, the strategy it depends if you have already some information about possible refolding buffer working from your specific proteinvor not.

If you already know from literature the refolding confitions, you can try to gradually remove urea by dialysis (eg performing suqsequntal step in 6M, 4M, 2M, 1M, no urea buffer) and at the end if you do not have protein precipitation, check the protein folding by DSF, CD or NMR or functional assays, if available.

If the protein is unknown and no refolding protocol are reported, i suggest to perform a preliminary DIRECT refolding screening approach.

You can concentrate the protein in urea and dilute directly it 10-20o times (to reach 10-20uM final) in a panel of buffers with different pH, ionic strength, and other agents (eg arginine), incubate O/N at 4°C and the following day check in with condition more protein is retained in solution. (after long centrifugation to remove the precipitate that at low concentration cold be not so visible)

You can check it or by determine the protein concentration in the surnatants or loading all the surnatants in SDS-page and in case you have some positive result, try to scale up.

You can work in 96 well plate and a small amount of protein is necessary.

There are some commercial buffer screening kits already avaialble, but ohterwise you can build your own buffer screening.

Did your protein contain Cysteines? Because the refolding of proteins with cisteines is much more difficult since you need to add in the buffer also dombinationo of reducing/oxidating agents.

good luck

Manuele

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