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Protein purification method selection and principles

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The purpose of protein purification is to separate the target protein from all the components of the cell lysate, while still retaining the biological activity and chemical integrity of the protein. The separation and purification of protein is an arduous and arduous task. It is necessary to select an appropriate purification method according to the characteristics of the protein to improve the purity of the obtained protein product.

The principle of protein purification is: different proteins have different amino acid sequences and spatial structures, leading to differences in their physical, chemical, and biological properties. Using the differences in properties between the protein to be separated and other proteins, a reasonable set can be designed Protein purification program. Protein purification can be roughly divided into two stages: coarse separation stage and fine purification stage. The coarse separation stage mainly separates the target protein from other cellular components such as RNA, DNA, etc. The commonly used method is ammonium sulfate precipitation. The purpose of the fine purification stage is to distinguish the target protein from other proteins with similar size and physical and chemical properties. Commonly used methods are: gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, etc.

Gel filtration chromatography

Gel filtration (also called size exclusion chromatography or molecular sieve) is a method of separating proteins from a mixture based on molecular size. There are differences in the shape and molecular size of different proteins. When the mixture passes through a gel filtration chromatography column containing packed particles, due to the different molecular sizes of various proteins, the ability to diffuse into particles of specific sizes and pores is also different. Large proteins The molecule will be eluted first, the smaller the molecule, the later it will elute, so as to achieve the purpose of separating the protein. Generally speaking, the thinner and longer the gel filtration chromatography column, the better the purification effect.

The molecular weight range of the protein that can be purified by gel filtration chromatography is wide, and the purification process does not require organic solvents that can cause protein denaturation. The disadvantage is that the sample load is usually only 5%-30% of the column volume, and it is difficult to pack the column, and the requirements for the chromatography column and the chromatography equipment are high; it is usually used in the protein fine purification stage.

Gel filtration chromatography
Gel filtration chromatography

Ion exchange chromatography

Ion exchange chromatography is a technique for separating and purifying proteins based on the amount of charge on the protein surface. The surface of the protein usually has a certain charge, and the charged amino acid residues are evenly distributed on the surface of the protein, and can be combined with the cation exchange column or the anion exchange column under certain conditions. The binding effect between this charged molecule and the stationary phase is reversible. When changing pH or eluting with a buffer that gradually increases ionic strength, the bound substance on the ion exchanger can be exchanged with the ions in the eluate. Is eluted into the solution. Due to the different charges of different substances, their binding ability with ion exchangers is also different, so the order of elution into the solution is also different, so as to achieve the effect of separation.

Ion exchange chromatography is the most widely used chromatographic technology. It is suitable for all stages of the separation and purification process. It can be used as a primary capture method or a fine purification method; it is characterized by large sample processing capacity and simple operation.

Affinity chromatography

Affinity chromatography purification is a technique for protein purification using the characteristics of biological macromolecules that have specific and reversible binding to certain corresponding molecules. this method

It is suitable for purifying the target from a mixture with complex components and far greater impurity content than the target. It has the advantages of good separation effect, mild separation conditions, high binding efficiency and fast separation speed. Affinity chromatography technology can use the specific adsorption between the ligand and the biomolecule to separate the protein, and it can also add a tag to the protein, and use the specific binding between the tag and the ligand to purify the protein.

Specific adsorption between ligands and biomolecules

Ligand: A specific substance that binds reversibly in affinity chromatography is called a ligand. Ligands can be enzyme substrates, inhibitors, cofactors, specific antibodies and the like.

Affinity chromatography is based on the difference in affinity between the ligands of the stationary phase and the biomolecules to separate each other. According to the selectivity of high and low affinity chromatography, it can be divided into group affinity chromatography and high selectivity (specific) affinity chromatography. Group affinity chromatography One ligand can adsorb multiple proteins, such as using triazine dye as a ligand to purify a class of proteins or glycoproteins containing glycosyl groups, and high-selectivity affinity chromatography can only adsorb one ligand The specific adsorption of a protein, such as a monoclonal antibody, to an antigen.

Using genetic engineering technology to add a few extra amino acids to the amino or carboxyl end of the protein, this added tag can be used as an effective purification

in accordance with:

GST tag purification: add glutathione S-transferase (GST) to the protein sequence, then use Qianchun Bio-GSH Purose 4 Fast Flow for affinity purification, and then use thrombin or factor Xa to cut;

His tag purification: His-tag is one of the most common tags. Add 6~10 histidines to the amino terminal of the protein. Using Ni-NTA Purose 6 Fast Flow affinity filler, it can The ability to bind tightly to the Ni2+ chelating column, eluting the target disc protein with imidazole.

Hydrophobic chromatography

Hydrophobic interaction chromatography is a chromatographic method that uses the difference in hydrophobic force between the hydrophobic group of the sample molecule in the salt-water system and the hydrophobic ligand of the chromatographic medium for separation. This method takes advantage of the hydrophobicity of the protein. The hydrophobic residues of the protein will be exposed on the surface of the protein after denaturation or in a high-salt environment. The hydrophobic residues of different proteins and the hydrophobic ligands of the stationary phase have different strengths. The high to low ionic strength eluent can be used to separate the hydrophobic components from weak to strong.

The hydrophobic interaction chromatography experiment has low operating cost and the purified protein has biological activity. It is a universal method for separating and purifying proteins. This experiment follows the principle of “high salt loading, low salt elution”: the protein in the high-concentration saline solution is retained on the column, and the protein is eluted from the column in the low-salt or aqueous solution. It is especially suitable for precipitation in concentrated ammonium sulfate solution. The separated mother liquor and the solution containing the target product after the precipitate is dissolved in salt are directly injected onto the column. Of course, the E. coli expressed protein extract solution of 7mol/guanidine hydrochloride or 8mol/L urea is also suitable for injection directly onto the column. Refolding was carried out at the same time of separation.

Hydrophobic chromatography is widely used and is often combined with other separation methods. For example, hydrophobic chromatography is very suitable for processing samples after salting out, or used in conjunction with ion exchange chromatography.

Tips: The basic principles of protein purification:

  1. The source of the unmaterial sample to be purified, the physical and chemical properties of the target protein must be fully understood (basically including molecular weight, PI, source of main contaminants, etc.);

  2. When designing the purification process, it is necessary to clarify the desired goals and objectives of the purification;

  3. According to the desired goal to be achieved, reasonable selection of factors such as purity, recovery rate, activity, experimental cost, etc., design a scientific and reasonable purification process;

  4. Familiar with the performance and function of various commonly used additives in protein purification process, reasonable addition and use, can double the result of the purification process with half the effort.

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