As the protein chemistry and molecular biology grow rapidly, the development and application of protein drugs for the treatment of various diseases has become a hot spot in biomedical industry. Polypeptides and protein drugs have mild adverse effects and strong activity while addressing both the symptoms and root cause. When we purify proteins, keeping the protein from inactivation and minimizing the loss of protein activity caused by the purification process are of primary significance. Hence, the following factors should be considered when designing buffer: pH , buffer system, salt ion, reducing agent and stabilizer.
pH
Many experiments set the pH at 7.4 to mimic biological conditions, but if the target protein is unstable under this condition, it is necessary to change the pH to make it soluble and non-degradable in the solution. When the pH of the solution is near the pI of the protein isoelectric point, it will not dissolve easily in the solution, because there is no net charge on the surface of the protein and protein can easily congregate. The ProtParam tool of ExPASy website can be used to calculate the protein isoelectric point pI value quickly and easily, as long as you submit protein sequence.
Buffer System
First, we need to ensure that the selected buffer system does have buffering ability at the set pH, and its dissociation constant pKa should be near the set pH within a unit.
The next step is to ensure that the buffer concentration is high enough to achieve the function of buffering. The usual concentration is 20~100 mM. It should be noted that the buffer system used can not affect protein activity. For instance, phosphate will inhibit activity of kinase, so it should be thoroughly dialyzed before the reaction.
Moreover, some buffer systems are very sensitive to the temperature, such as Tris-HCl buffer. If the buffer system is set to pH 8 at 25℃, its pH will increase to 8.58 at 5℃ and decrease to 7.71 at 37℃. Therefore, if the experiment is not under the condition of 25℃, it should be considered that this pH may not be applicable under the experimental conditions.
Salt Ions
Many buffers contain NaCl to help maintain protein solubility and mimic physiological conditions with the concentration of 150 mM, but different salt ion concentrations may be required in different protein purification steps. For ion exchange chromatography, you usually bind in low salt and elute in high salt. Reducing salt concentration when binding is to avoid salt ions compete with protein compete for binding packing materials, and to prevent protein from flowing through the ion exchange column so that the column can bind the target proteins under high ionic strength; While hydrophobic chromatography generally includes high salt binding and low salt elution. For hydrophobic chromatography, you usually bind in high salt and elute in low salt.
Reducing Agent
If the target protein contains cysteine residues, there may be oxidation problems between residues and lead to protein aggregation. to prevent this, some reducing agents such as DTT、TCEP and mercaptoethanol are often added to the buffer.
TCEP is the most stable but also the most expensive of the three reducing agents. We usually add DTT to the buffer during purification process, then add TCEP to the buffer that used to reserve the enzyme liquid. The reducing agent concentration is generally 5~10 mM, which should be much higher than the protein concentration. DTT and mercaptoethanol will degrade at room temperature, so it is necessary to keep the buffer added reducing agent at low temperature or add reducing agent when using.
When using reducing agent, make sure that the column materials can be compatible with it. For example, the high concentration of reducing agent will take off the nickel in the Ni column and make the the column brown. Although the Ni column can be regenerated, the column loading capacity will be greatly affected under this specific condition.
Stabilizer
Adding some stabilizers to the buffer can help to improve the protein solubility and stability during protein purification. Adding inert protein to the buffer BSA can stabilize the target protein to some extent, but we must ensure that these added stabilizers do not interfere with the experiment. Sometimes, glycerol, polyethylene glycol and other substances will be added to increase buffer viscosity and prevent protein aggregation. In addition, using small amount of surfactants and some ionic compounds such as sulfate, amino acid, citric acid can avoid ionic interactions between proteins or help protein dissolve.
These are the five factors that should be considered in choosing buffer. In order to maintain the activity and stability of the protein during purification, it is recommened to design the best experimental scheme.
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