With the development of chemistry and molecular biology in protein, the research and application of protein Prize drugs for the treatment of various diseases has become a hot spot in the development of biomedical industry. And polypeptide and protein drugs have little side effect, strong activity and the effect of treating both symptoms and root causes. When we purify protein, the most important thing is to keep the protein not inactivated during the purification process, or to minimize the loss of protein activity caused by the purification process. Therefore, the following factors should be considered when designing buffer: pH value, buffer system, salt ions, reducing agent and stabilizer.
In many experiments, the pH value is set at 7.4 to imitate biological conditions, but if the target protein is unstable under this condition, it is necessary to change the pH value to make it soluble in solution and not degradable. When the pH value of the solution is near the isoelectric point pl of protein, it will be difficult to dissolve in the solution, because there is no net charge on the protein surface at this time, so it is easy to aggregate. You can use ProtParam tool of ExPASy website to calculate the pl value of protein isoelectric point quickly and easily, as long as you submit the protein sequence.
First of all, we should ensure that the selected buffer system does have buffer capacity at the set pH value, and its dissociation constant pKa value should be within one unit above and below the set pH value. Furthermore, it is to ensure that the concentration of buffer solution is high enough to achieve the function of actual buffer solution. Generally, the chosen concentration is 20~100mM. It should be noted that the buffer system used cannot affect the activity of protein, for example, phosphate can inhibit the activity of kinase, so it should be thoroughly dialyzed before reaction.
In addition, some buffer systems are very sensitive to temperature, such as Tris-HCL buffer. If the buffer system is adjusted 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 conducted at 25℃, it should be considered that this pH may not be applicable in the experimental conditions.
Many buffers contain NaCl to help maintain the solubility of protein and simulate physiological conditions. The concentration is generally 150mM, but different salt ion concentrations may be needed in different protein purification steps. Ion exchange chromatography is generally characterized by low-salt binding and high-salt elution. When binding, it is necessary to reduce the salt concentration in order to prevent salt ions from competing with proteins to bind with fillers under high ionic strength, and to prevent proteins from flowing through the ion exchange column, so that the column can bind the target proteins. Hydrophobic chromatography is generally characterized by high salt binding and low salt elution.
If the target protein contains cysteine residues, oxidation between residues may occur, which may lead to protein aggregation. To prevent this, some reducing agents, such as DTT, TCEP and thioglycolysis, are often added to the buffer.
TCEP is the most stable of the three reducing agents, but it is also the most expensive. Usually, DTT will be added to the buffer in the purification process, and TCEP will be added to the buffer in which the enzyme solution is finally stored. The concentration of reducing agent is generally 5~10mM, which should be much higher than that of daughter protein. DT and thioglycolysis will degrade at room temperature, so it is necessary to store the buffer with reducing agent at low temperature, or add reducing agent when using.
When using reductant, it is necessary to ensure that the material of the column can be compatible with it. For example, a high concentration of reductant will strip off the silver in the nickel column and make the color of the column dark and brown. Although the nickel column can be regenerated, the column load will be greatly affected.
Adding some stabilizers to the buffer solution can help improve the solubility and stability of protein during protein purification. Adding emotional protein BSA to the buffer can stabilize the target protein to some extent, but it must be ensured that these added stabilizers do not interfere with the experiment; sometimes adding glycerol, polyethylene glycol, etc. to increase the viscosity of the buffer helps to prevent protein aggregation; in addition, using a small amount of surfactant and some ionic compounds such as sulfate, amino acids, citric acid, etc. can avoid the ionic interaction between proteins and help protein dissolution.
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