To extract protein from inclusion bodies, you must first lyse cells to release them and then solubilize the inclusion bodies using strong denaturing agents like guanidine HCl or urea to break apart the aggregated protein. After solubilization, you must refold the protein by slowly removing the denaturant, often through dilution or dialysis, to restore its functional, native structure and then perform subsequent purification steps.
Step-by-Step Process
- Cell Lysis:
- Disrupt bacterial cells using mechanical methods such as sonication or high-pressure homogenization, or enzymatic methods with lysozyme.
- Inclusion Body Isolation:
- Centrifuge the cell lysate to pellet the insoluble inclusion bodies.
- Wash the pellet multiple times with a buffer to remove soluble cellular debris, including proteases.
- Solubilization:
- Resuspend the inclusion body pellet in a solution containing a strong chaotropic agent (e.g., 8 M urea or 6 M guanidine hydrochloride).
- Add a reducing agent (such as dithiothreitol or β-mercaptoethanol) to break disulfide bonds within the protein aggregates.
- Mechanical dispersion (sonication, stirring) may be needed for complete dissolution.
- Refolding:
- Slowly remove the denaturing agent to allow the protein to refold into its native, active structure.
- Methods include gradual dilution into refolding buffer or dialysis against a series of decreasing denaturant concentrations.
- Purification:
- Once the protein is refolded, it can be purified using appropriate chromatography techniques, such as affinity chromatography (if a tag is present) or ion-exchange chromatography, to remove remaining impurities.