Refolding inclusion bodies involves solubilizing the aggregated protein and then inducing it to fold correctly into its functional form. 

General Steps for Refolding Inclusion Bodies:

     1.     Isolation of Inclusion Bodies: 

          ●      Process: Lyse the cells to release the inclusion bodies and then separate them from the soluble proteins using centrifugation.

          ●      Purpose: Obtain a pure preparation of inclusion bodies for further processing.

     2.     Solubilization: 

          ●      Process: Dissolve the inclusion bodies in a strong denaturant solution, such as urea or guanidine hydrochloride, often with the addition of reducing agents (e.g., dithiothreitol, DTT) to break disulfide bonds.

         ●       Purpose: Solubilize the aggregated protein and disrupt the aggregates into individual polypeptide chains.

     3.     Refolding: 

         ●       Process: Gradually remove the denaturant by dilution or dialysis against a refolding buffer containing refolding aids, such as:

               ●      Buffer: A buffer with low concentrations of denaturant, salts, and often additives like glycerol or arginine.

               ●      Redox Agents: To facilitate the formation of correct disulfide bonds (e.g., oxidized and reduced glutathione).

               ●      Chaperones: Optional addition of molecular chaperones or protein disulfide isomerase (PDI) to assist in correct folding. 

          ●      Purpose: Allow the protein to refold into its native conformation.

     4.     Purification: 

          ●      Process: Purify the refolded protein using chromatographic techniques such as affinity chromatography, ion exchange chromatography, or size-exclusion chromatography.

          ●      Purpose: Remove any remaining aggregates or impurities and obtain the properly folded, functional protein.

     5.     Characterization: 

          ●      Process: Assess the refolded protein for correct folding and functionality using techniques such as SDS-PAGE, Western blotting, activity assays, or circular dichroism (CD) spectroscopy.

          ●      Purpose: Ensure that the refolded protein is in its active form and meets quality standards.

Tips for Successful Refolding:

          ●      Optimize Refolding Conditions: The concentration of denaturant, refolding buffer composition, and refolding temperature should be optimized based on the specific protein.

          ●      Gradual Denaturant Removal: Avoid rapid removal of denaturants to prevent the formation of new aggregates. 

          ●      Additives: Use additives that can stabilize intermediate folding states and prevent aggregation during refolding.

Refolding inclusion bodies can be complex and may require multiple optimization steps to achieve high yields of functional protein.

GenCefe provide one-stop solutions from codon optimization, gene synthesis, to protein expression and purification. In addition to four conventional expression systems of bacterial, yeast, insect cell, and mammalian cell, we have also developed a patented cell free expression technology, which is suitable for the production of difficult proteins such as toxic proteins and membrane proteins.