2022-02-15 10:42:43
Proteins are typically present in complex mixtures in tissues or cells and therefore require purification in many experimental applications such as structural studies and in vitro biochemical assays. Protein purification is based on their different physical properties to separate from the raw materials in order to achieve the purpose of obtaining the most functional proteins. Among them, the protein-purified solution has played a crucial role, then what ingredients are needed in the solution?
Buffer system
Proteins should be kept in a good buffer environment to prevent irreversible effects on protein folding status, solubility and activity. Biochemical experiments commonly used pH value of 7.4, but in protein purification experiments, the protein isoelectric point also should be considered. At the isoelectric point, the protein molecules exist in the form of zwitterions with equal positive and negative charges and zero net charge. Their particles can easily collide and aggregate to form precipitates. Therefore, the pH should be set according to the isoelectric point of the protein. A good buffer must have the following characteristics:
Good compatibility with other solutes.
Common buffer system are shown in the following table:
Buffer System |
pH |
Advantages |
Disadvantages |
Tris |
7.5~9.0 |
The preparation process is simple; little interferes with the system |
pH changes with temperature or dilution |
Phosphoric acid |
5.8~8.0 |
pH does not change with temperature |
Easier to form precipitates with common Ca2+, Mg2+, and heavy metal ions; inhibit certain biochemical processes and most enzyme activities |
MOPS |
6.5~7.9 |
High buffering capacity at physiological pH range |
Can not be autoclaved;The pH changes to a certain extent with temperature
|
HEPES |
6.8~8.2 |
high solubility, does not affect the chemical reaction, little environmental impact |
Can not be autoclaved; pH changes to a certain extent with temperature; will generate free radicals under certain conditions |
Reducing agent
If the protein contains cysteine residues, oxidation between cysteine residues may cause protein aggregation. In this case, it is often necessary to add a reducing agent to the buffer. Commonly used reducing agents are β-mercaptoethanol (BME), dithiothreitol (DTT) and tris-(2-formyl ethyl) phosphine hydrochloride (TCEP-HCl). BME contains one mercapto group, which has the worst reducibility and can only maintain 2~3 days. DTT has two mercapto groups, which is stronger than BME for 3~7 days. The reduction ability of TCEP is stronger than that of BME and DTT, its role can be maintained for 2~3 weeks. TCEP not only has strong reduction ability, high selectivety, but also more stable both in acidic or alkaline conditions.
Protease inhibitor
Protease inhibitors in a broad sense, refers to the materials that can combine with the active site of protease molecules, so that protease activity decreased or even disappear, but not to denature the enzyme protein. It is often added in the early steps of lysis buffer and purification process to prevent the enzymolysis of target protein by endogenous proteases. Common protease inhibitors are:
Leupeptin—serine and cysteine protease inhibitors;
Gastric Inhibitory Polypeptide—aspartic protease inhibitor;
PMSF—serine protease inhibitors
There are other additives, such as the metal chelator EDTA or EGTA, which can chelate Ca2+ or Mg2+ to prevent the target protein from being degraded by the metalloprotease; arginine kinases that stabilize the protein structure and enhance solubility; ionic stabilizers, enhance solubility, and so on.
Protein separation and purification is a complex and important work, which directly impact on the success of subsequent experiments. However, there is no single or a set of ready-made methods to extract any one protein from a complex mixture, so in practice it is also necessary for the experimenter to explore the conditions to choose the most appropriate way to obtain high-purity products.
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