As an optional service in our bacterial protein expression platform, the in vitro denaturation and refolding of inclusion bodies service is now available at Creative BioMart.
In the cell, there is competition between folding and aggregations. In many cases and in several host systems, recombinant proteins accumulate intracellularly in insoluble aggregates. The proteins in these so-called inclusion bodies are mostly inactive and denatured. In addition, dimers and multimers may be present. However, the expression of recombinant proteins in inclusion bodies can also be advantageous:
The major problem is to recover biologically active and/or soluble protein in high yield.
In order to accomplish this, the protein in the inclusion bodies must be solubilized and refolded in vitro. This procedure is carried out in 2 phases:
1. Isolation of inclusion bodies
Inclusion bodies have a relatively high density and, therefore, can be pelleted by centrifugation. Cells are usually disrupted by high-pressure homogenization (optionally following a lysosyme treatment). It is important that cell lysis is complete, because intact cells sediment together with the inclusion bodies, thus contaminating the preparation. After centrifugation, the pellet is washed with buffer containing either low concentrations of chaotropic agents or detergents. This wash step is necessary to remove contaminants, especially proteins (proteases), that may have been absorbed onto the hydrophobic inclusion bodies during processing.
2. Solubilization of aggregated proteins
The washed inclusion bodies are resuspended and incubated in a buffer containing a strong denaturant and a reducing agent (usually 20 mM DTT or b-mercaptoethanol). The addition of a reducing agent keeps all cysteines in a reduced state and cleaves disulfide bonds formed during the preparation. Incubation temperatures above 30°C are typically used to facilitate the solubilization process. Optimal conditions for solubilization are protein-specific and have to be determined for each protein. This is done most effectively by carrying out small-scale experiments (1-2 ml) to screen the different variables.
Refolding of the solubilized proteins
Refolding of the solubilized proteins is initiated by the removal of the denaturant. The efficiency of refolding depends on the competition between correct folding and aggregation. To slow down the aggregation process, refolding is usually carried out at low protein concentrations, in the range of 10-100 mg/ml. Furthermore, refolding conditions must be optimized for each protein.
Different methods for the refolding of proteins we offer:
| REFOLDING METHOD | DESCRIPTION |
| Dialysis | The most used method is the removal of the solubilizing agent by dialysis |
| Slow dilution | The concentration of the solubilizing agent is decreased by dilution allowing the protein to refold. Usually, the dilution is carried out slowly by step-wise addition of buffer or by continuous addition using a pump. |
| Pulse renaturation | In order to keep the concentration of the unfolded protein low, thus limiting aggregation, aliquots of denatured protein are added at defined time points to the refolding buffer. |
Customers who need our assistance are more than welcome to contact us or submit an online inquiry. Our professional team will provide you with detailed guidance and solution ASAP. If you have any special needs or be interested in learning more about Creative BioMart's protein expression services, please feel free to inquiry for more details.
