The Protein Man's Blog | A Discussion of Protein Research

Maintaining the stability and solubility of target proteins before, during and after purification is one of the biggest challenges researchers often run into. Proteins are extremely sensitive to solution conditions (i.e., pH and conductivity) and temperature. They also tend to aggregate at high concentrations required for structural studies. This can be a serious problem since protein aggregation can cause artifacts and hamper the biological activity of the target protein.

Protein aggregation can be detected in a number of ways. In some cases, the particulate matter can be visually observed in the solution while very large particles can sometimes be detected in the void-volume during size exclusion chromatography and/or during Dynamic Light Scattering. The presence of protein aggregates can also be inferred from loss of activity and/or the presence of experimental artifacts.    

Preventing Protein Aggregation: 5 Useful Tips to Consider

There are several ways by which you can prevent and/or overcome aggregation when working with your protein of interest. Consider these helpful tips.

Maintain low protein concentration. High protein concentrations compromise the stability of your target protein so increasing sample volume during lysis and chromatography can be a good idea. If you need a high final protein concentration, consider adding stabilizing buffer components.

Work at the right temperature. Purified proteins are generally unstable at 4^oC so store them at -80^oC and use a good cryoprotectant (e.g., glycerol) to prevent aggregation during freeze-thaw cycles.

Change the pH of the solution. Most experiments are conducted at pH 7.4, but if your target protein is not stable at this pH, you need to find the right condition that will keep your protein in the solution. Since proteins are least soluble when the pH of the buffer used is equal to the pl of the protein (i.e., the net charge on the protein = 0), changing the pH of the current buffer will charge the net charge of the protein and modulate the interactions that may lead to aggregation.

For example, you can consider raising the pH by 1 unit if the pl is less than the buffer pH. Alternatively, you can lower the pH by 1 unit if the pl is greater than the buffer pH. However, try to maintain the ionic strength of the buffer when making significant changes to the pH to avoid erroneous results.

Change the salt concentration. The electrostatic interactions within and between protein molecules can be affected by the ionic strength of the buffer, so try to determine the optimal buffering environment for your target protein. Alternatively, try using different salts to achieve protein stability and prevent aggregation.

Use an appropriate additive. Certain additives such as those mentioned below can help optimize buffer conditions and improve protein solubility during purification.

• Since osmolytes (e.g., glycerol, sucrose, TMAO (Trimethylamine N-oxide)) interact with the exposed amide backbones of proteins, their presence exerts a stabilizing effect and prevents aggregation. Osmolytes are known to favor proteins in the native state (with less-exposed backbones) over those in the denatured state (with more exposed backbones).
• Amino acids. Adding a mixture of arginine and glutamate to your buffer increases protein solubility by directly binding to charged and hydrophobic regions.
• Reducing agent. For target proteins containing cysteine residues, consider adding a reducing agent (e.g., ß-mercaptoethanol, DTT and TCEP (tris 2-carboxyethyl phosphine)) to help prevent oxidation and protein aggregation. Note: Keep BME and DTT in the refrigerator since they tend to break down at room temperature. Also, consider adding the reducing agent when you’re about to use the buffer.
• Non-denaturing detergent. Adding low concentrations of non-denaturing detergents help solubilize protein aggregates without denaturing the proteins. For best results, use non-ionic or zwitterionic detergents (e.g., Tween 20, CHAPS).
• Adding a ligand to the protein solution before concentration will favor the population of proteins in their native state and reduce the exposure of hydrophobic patches in ligand-binding sites to prevent aggregation. Non-detergent sulphobetaines can also be used to achieve the same effect.