Several problems should be paid attention to in the process of protein purification. Firstly, the pH value should always be controlled in the purification process to avoid the irreversible change of protein conformation caused by over acid or over alkali. The buffer should have a suitable pH value and be harmless to proteins. When purifying proteins in plants and fungi, a large buffer capacity is usually required. At this time, pay attention to the concentration of the buffer, whether the components of the buffer are appropriate and whether there will be side reactions. Proteins containing sulfhydryl groups (in which sulfhydryl groups are not bound into disulfide bonds) are particularly easy to be oxidized. Therefore, a reducing environment should be maintained. Some reducing agents containing sulfhydryl groups can be added to keep the whole system in an oxygen free state to solve this problem, but in some cases, the added sulfhydryl compounds may inhibit the activity of proteins.

During the purification process, it is usually necessary to keep low temperature, because there is protein hydrolase in the cell, which is activated after tissue homogenization and can degrade protein. Therefore, low temperature must be maintained to reduce the role of hydrolases. However, it should be noted that sometimes low temperature can destroy the quaternary structure of some enzymes. Generally, protein is stable at 0 ℃, but acetone carboxylase is sensitive to cold and stable at 25 ℃. Some enzymes need to be stable at – 20 ℃ or – 70 ℃.

One of the biggest problems in the process of protein purification is that in the whole process of protein purification, the protein is more or less always affected by the solution environment which is very different from the physiological state. This is very noteworthy for proteins whose function is highly controlled in living cells. On the contrary, some proteins, such as secretory proteins, can adapt to large environmental changes without changing their structure and function. In order to reduce the spontaneous denaturation of protein in dilute aqueous solution, some small molecular weight substances (such as sucrose, glycerol, etc.) or other proteins (such as bovine serum albumin, gelatin, etc.) can be added to the protein solution to stabilize the protein. This is a method to imitate the intracellular environment and reduce the harmful effect of water. This is also the reason why albumin is added as a stabilizer in many gene protein drugs. Sometimes, dimethyl sulfoxide and dimethylformamide are even added, and the added concentration needs to be tested, generally 1% – 10%. A few proteins can maintain their activity in polar media with high ionic strength, such as KCl, NaCl, (NH4) 2SO4, etc.

In addition, some commonly used methods can effectively reduce protein denaturation. For example, the existence of some divalent ions (Mg2 +, Ca2 +, etc.) contributes to the stability of proteins or protein complexes; When purifying enzyme, specific substrate can be added; Sometimes it is necessary to use complexing agents (such as ethylenediamine tetraacetate) to remove unwanted ions, especially those harmful metal ions brought by water sources. These methods can be used in different situations.

Another factor to note is that even if you are careful in the purification process, the protein will change structurally. Therefore, the conformation of purified protein may be different from natural conformation or fully functional conformation even if its vitality is basically retained. In this case, the properties of proteins measured outside the organism may not accurately reflect the properties of these macromolecules in vivo.

The dissociation of proteins containing multiple subunits is a special type of denaturation. When purifying proteins, when the measured activity is expressed by only one subunit, other subunits may be lost because they are not noticed. Some proteins have multifunctional forms in vivo, and their biological activity is displayed by proteins determined by more than two different genetic genes. Therefore, special care should be taken when inferring the complete biological function of a purified protein in vivo from its properties. At this time, it is beneficial to analyze the total activity in the purification process. In the early stage of purification, if the total activity is used as the benchmark, it is expected to recover 50% of the total activity at the end of purification, but the actual yield is often only 5% – 20%. In some cases, especially when a large number of raw materials are available, we can concentrate on obtaining samples with high specific activity without worrying about the yield.

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