Refolding of Inclusion Bodies

Expression

Grow cells to OD 600 of 0.7–0.8
Add 1 mM IPTG at 37 oC for 4 hours.
Spin down cells
lyse cell with Lysis Buffer and spin.

50mM Tris-HCl, pH 8.0
100mM NaCl
5mM EDTA
0.5% Triton-X100

1.6 M Urea
1 mM b-mercaptoethanol

Add IB Washing Buffer to pellet

• 50 mM Tris, 50 mM NaCl,  1.5 mM b-mercaptoethanol,  pH 8.0
  

Wash the inclusion bodies two  times

Spin and remove supernatant.

One step denaturing

Dissolve inclusion bodies in 5 ml of IB Extraction Buffer

• 50 mM Tris, 50 mM NaCl, 10 mM b-mercaptoethanol, 8 M urea, pH 8.0

Stire the solution for about 20 minutes
Spin at 25000 rpm for 30 min at 4 oC.
Collect the supernatant, and discard the pellets.
Adjust protein concentration to 8–10 mg/ml.

Two-step denaturing

Dissolve inclusion bodies in 5 ml of IB Extraction Buffer II

• 50 mM Tris, 50 mM NaCl, 10 mM b-mercaptoethanol, 7 M GdnHCl, pH 8.0

Stire the solution for about 20 minutes
Spin at 25000 rpm for 30 min at 4 oC.
Collect the supernatant, and discard the pellets.
Add the supernatant rapidly into 200 ml of IB Dilution Buffer

• 50 mM Tris, 1 mM EDTA, 50 mM NaCl, 10 mM b-mercaptoethanol, pH 8.0 to precipitate protein

Spin at 25000 rpm for 30 min at 4 oC.
Remove supernatant and dissolve pellets in 5 ml of IB Extraction Buffer II

• 50 mM Tris-HCl, 50 mM NaCl, 10 mM b-mercaptoethanol, 8 M urea, pH 8.0

Adjust protein concentration to 8–10 mg/ml.

Refolding of inclusion bodies

Add protein solution dropwise into 400 ml of IB Refolding Buffer

• 20 mM Tris, 1 mM EDTA, 1 mM GSH, 0.1 mM GSSG, pH 8.0

Stir slowly with magnetic stir bar at 4 oC for two days
Load protein at a rate of 0.5 ml/min onto a Q or SP column equilibrate with refolding buffer.
Elute protein using a linear salt gradient from 50 mM–1 M NaCl in a buffer

Refolding by Dialysis

Refolding buffers (RF)

• 50 mM Tris-HCl, pH8.0,  0.4M L-Arginine, 1 mM EDTA

supplement with 4.0M, 2.0M, 1.0M and 0.00M Urea. (4M urea may require heat as well as
vigorous stirring to dissolve)

step 1. dialyze against RF buffer with 4.0M urea for 24hrs
step 2. dialyze against RF buffer with 2.0M urea for 24hrs
step 3. dialyze against RF buffer with 1.0M urea for 24hrs
step 4. dialyze against RF buffer with 0.0M urea for 24hrs
step 5. dilute 0M refolding buffer with water in a 1:4 ratio, dialyze against 1/4*RF buffer for 24hrs
step 6. dialyze against a buffer suitable for next step purification.
Some steps can continue longer than 1 day if necessary.

optionally, replace 8M Urea with 10% glycerol +6M guanidinium-Cl

Refolding with GSH/GSSH and Arginine

Dissolve the inclusion body pellet in resuspension buffer
50 mM Tris, pH 8, 6 M GuHCl, 10 mM DTT) by repeatedly passing the inclusion bodies through an 18g syringe.
Remove insoluble material by centrifugtation.
Dilute the resuspended protein material to 50% in dialysis buffer #1 (50 mM Tris, pH 8, 2 M GuHCl) resulting in a 4 M GuHCl containing solution.
Dialyse the protein solution at 4 oC in a dialysis tubing against 2 L of buffer #1 o/n.
Replace with 2 L of dialysis buffer #2
50 mM Tris, pH 8, 1 M GuHCl, 0.4 M Arginine, 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione.
Dialyse overnight at 4 oC.
Dilute the dialysis buffer with equal volume of water
dialysis overnight.
Spin to remove any insoluble material.
Dialyse the remaining protein solution overnight at 4 oC against 1 L of dialysis buffer #3
(50 mM Tris, pH 8, 250 mM NaCl, 0.1 M Arginine, 3 mM Reduced Glutathione, 0.9 mM Oxidized Glutathione) to remove the residual GuHCl.

On-column Refolding

Washing

Resuspend inclusion pellet in 3 ml cold 2 M urea, 20 mM Tris-HCl, 0.5 M NaCl, 2% Triton TM X-100 pH 8.0

Sonicate

Spin at high speed for 10 min. at + 4 °C.

Subject the pellet to a second round of urea wash.

Wash the pellet once in buffer lacking urea and store frozen for later processing.

Solubilization

Resuspend the pellet in 5 ml 20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM
2-mercaptoethanol pH 8.0.

Stir for 30–60 min. at room temperature and centrifugate 15 min. at high speed, + 4 °C.
Remove remaining particles by passing the sample through a
0.22 μm or 0.45 μm filter.
The optimal concentration of reducing 2-mercaptoethanol (0–5 mM) must be determined experimentally for each individual protein.

Load to 5~10 ml Ni column

Load the sample

wash the column with 1 vol of  20 mM Tris-HCl, 0.5 M NaCl, 5 mM imidazole, 6 M guanidine hydrochloride, 1 mM 2-mercaptoethanol pH 8.0.

Wash column with 1 vol of  20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole,  1 mM 2-mercaptoethanol, 6 M urea, pH 8.0

Folding

Refolding of the bound protein is performed by the use of a linear 6–>0 M urea gradient, starting with the wash buffer above and finishing at one without urea. A gradient volume of 30 CV or higher and a flow rate of 0.5–1 ml/min can be used, while the optimal renaturation rate should be determined experimentally for each protein. Continue to
wash with 5 ml of buffer without urea after the gradient has come to its endpoint

Elution

10 CV linear gradient starting with 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0. and ending with the same buffer including 500 mM imidazole

Refolding chemicals

reduced oxidized glutathione (GSH/GSSH), DTT/GSSH, cysteine/cystine, and cysteamine/cystamine at a total concentration of 5–15 mM with a molar ratio of reduced to oxidized compounds of 1 : 1 to 5 : 1, respectively

GSSG is oxidized glutathione, GSSH reduced glutathione.

Other chemicals

β-cyclodextrin