Both during the purification process of recombinant proteins and during their subsequent use in different techniques, maintaining the stability and solubility of the protein in question is a challenge for the researchers.
In this post we will see some factors that directly affect the solubility of proteins, and how to act on them to prevent their aggregation and precipitation and avoid the formation of inclusion bodies .
Below we list the 5 main factors to take into account in order to prevent aggregation phenomena when working with proteins, and some tips to avoid the formation of inclusion bodies.
1.- PROTEIN CONCENTRATION
Normally, when working with high protein concentrations, they tend to lose stability and increase the risk of precipitation. In these cases, the use of stabilizing additives is recommended.
Generally, the purified proteins are unstable at 4 ° C, -80 ° C so stored together recommended with the use of cryoprotectants to prevent aggregation during cycles of freeze / thaw .
As a rule, proteins are less soluble when the pH of the buffer is equal to the isoelectric point (pI) of the protein. Therefore, modifying the pH of the buffer will mean changing the net charge of the protein and thus modulating the interactions that may lead to the formation of aggregates, increasing its solubility.
4.- CONCENTRATION OF SALTS
The ionic strength of the buffer directly affects the electrostatic interactions between the proteins, so it will be necessary to optimize the concentration of salts in the buffer to avoid their precipitation.
The use of certain additives can improve the solubility of proteins:
They interact with exposed amide groups, promoting stability to avoid the formation of inclusion bodies.
- Amino acids
Solubility can also be improved by adding a mixture of arginine and glutamate that will bind to the shit and hydrophobic regions of the protein improving its stability.
- Reducing agents
The addition of some reducing agent such as β-mercaptoethanes or DTT, prevents oxidation and aggregation phenomena in those proteins that contain cysteine residues on the surface.
- Non-denaturing detergents
At low concentrations, these detergents help to re-solubilize protein aggregates without denaturing them.