Bacterial inclusion bodies are nightmare when we develop recombinant protein.
The first step, which generally is used, is dispersing the inclusion bodies in a denaturant such as 6M guanidine HCl (GuCl). The GuCl yields a solution of unfolded and essentially noninteracting polypeptide chains. The challenging steps are the protein is recovered in its unique, functional, and three dimensional conformation. Next, containing disulfide bonds in the native state is to understand how to renature the recombinant proteins. Reduction of disulfide bond resulting in unfolding and aggregation even in the absence of denaturant.
How to fold proteins are a key factor to obtain active form of the recombinant proteins. The three factors must be considered.
- The folded protein must have the most stable state.
- Kinetic barriers in the folding pathway must be minimized.
- Intermolecular aggregation must be reduced as much as we can.
The GuCl denaturation has a reversible unfolding transition depending on the concentration. The GuCl has 2.5-fold more effectiveness than urea which is considered milder denaturation agents, on a molar basis. However urea makes more stable intermediate status against aggregation than GuCl. Hence, urea is occasionally used as an intermadiate solvent during renaturation.
Cosolvents such as glycerol, ethylene glycol, glucose, sucrose, phosphate, sulfate, and 2-(N-morpholino) ethanesulfonic acid (MES) can stabilize proteins against denaturation by urea or GuCl. These agents work stabilizing hydrophobic interactions and aggregations (1).