This protocol is modified from Handling Inclusion Bodies in Recombinant Protein Expression and Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli (Palmer, I., & Wingfield, P. T. (1995). Preparation and Extraction of Insoluble (Inclusion-Body) Proteins fromEscherichia coli. Current Protocols in Protein Science. doi:10.1002/0471140864.ps0603s00 )
Isolation of inclusion bodies
Place thawed E. coli cells in a beaker. Add 10 ml lysis buffer per gram wet weight of cells. Keep bacterial cells cool by placing the beaker on ice in an ice bucket .
Sonicate the cell suspension for 5 min.
Clarify the lysed cell suspension by centrifugation for 15 min at 12,000 rpm, 4C.
Transfer the supernatant to the new test tube and purify your protein using NiNTA as usual. If your protein is insoluble, then go to step 5.
Add 5 ml Wash buffer per gram wet weight of cells, suspend the pellet with a glass rod or equivalent, and sonicate for 5 min.
Centrifuge the suspension for 10 min at 12,000 rpm, 4 C, and discard the supernatant.
Repeat step 5 one twice.
Dissolve the pellet with wash buffer containing 8 M urea, 10 mM DTT for 60 min and centrifuge as above.
Collect the supernatant.
Protein refolding was achieved by dialyzing the solution against a 1-L solution containing 0.1 mol/L Tris–HCL (pH 8.0), 5 mmol/L EDTA, and 5 mmol/L cysteine (4 °C, 16 h). The solution was centrifuged (10,000 rpm × 10 min) to eliminate precipitated proteins and purified with His Bind resin chromatography (Novagen) based on the manufacturer′s instructions.
For recombinant protein refolding, 1 ml of elution fractions were collected and pooled fractions were dialyzed overnight at 4 °C against 20 mM sodium phosphate buffer (pH 5.8). The dialyzed protein sample was then centrifuged at 13,000 rpm for 20 min at 4 °C to remove unfolded or aggregated proteins and analyzed by SDS-PAGE. The concentration of rSWDPm2 protein was determined by Bradford's method, and its molecular weight estimated by coresolution with standard markers through a 15% (w/v) SDS-PAGE gel.
Briefly, the dissolved inclusion bodies (1 mg/ml) were dialyzed continuously overnight at 4 °C against dialysis buffer containing 3, 2, 1, and 0 M urea and 0.01 M PBS. Oxidized glutathione and L-arginine were both added into the dialysis buffer containing 0.5 and 1 M urea.