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Principle of protein purification method

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The reason why a protein can be purified from a mixture of thousands of proteins is that different proteins have great differences in many of their physical, chemical, and biological properties. These properties are due to the sequence and number of amino acids in the protein. Due to the difference, the amino acid residues connected to the polypeptide backbone can be positively charged, negatively charged, polar or non-polar, hydrophilic or hydrophobic. In addition, the polypeptide can be folded into a very definite secondary structure (Α-helix, β-sheet and various turning angles), tertiary structure and quaternary structure, forming a unique size, shape and distribution of residues on the protein surface, using the difference in properties between the protein to be separated and other proteins , That is, a set of reasonable fractionation steps can be designed. The protein mixture can be separated according to the method corresponding to the different properties of the protein.

Different properties of the protein
Different properties of the protein

Molecular size

Different types of proteins have certain differences in molecular size, and some simple methods can be used to separate protein mixtures.

1.1 Dialysis and ultrafiltration

Dialysis is very commonly used in purification and can remove salts (desalting and replacement buffers), organic solvents, low molecular weight inhibitors, etc. Ultrafiltration is generally used to concentrate and replace solutions.

1.2, centrifugal separation replacement buffer

Many enzymes are enriched in a certain organelle. After homogenization, a certain subcellular component is obtained by centrifugation, so that the enzyme is enriched by 10-20 times, and then the specific enzyme is purified. Differential centrifugation has low resolution and is only suitable for rough extraction or concentration. Rate zone method, if the centrifugation time is too long, all the substances will be precipitated, so it is necessary to select the best separation time to obtain fairly pure subcellular components for further purification, avoiding the precipitation of large and small components together in differential centrifugation Problem, but the capacity is small and can only be used for small quantities. The commonly used media for isocratic gradient centrifugation are sucrose, polysucrose, cesium chloride, potassium bromide, and sodium iodide.

1.3, gel filtration (GF)

This is one of the most effective methods for separating protein mixtures based on their molecular size. Take care that the molecular weight of the protein to be separated falls within the working range of the gel. Choosing different molecular weight gels can be used for desalting, replacing buffers, and using molecular weight differences to remove heat sources.


Proteins are affected by their shape as they move through the solution during centrifugation, or move through membranes, gel filter particles, or pores in electrophoresis gels. For two proteins of the same mass, the globular protein has a smaller effective radius (Stokes radius), and the friction force encountered when settling through the solution is small, and the settling is faster and appears larger than other shapes of proteins; on the contrary, In size exclusion chromatography, globular proteins with a smaller Stoke radius are more likely to diffuse into the inside of the gel filtration packing particles and elute later, so they appear smaller than proteins of other shapes.


A common method for separating various proteins using the difference in protein solubility. There are many external factors that affect protein solubility, among which are: pH, ionic strength, dielectric constant and temperature of the solution. However, under the same specific external conditions, different proteins have different solubility. Appropriately change the external conditions to control the solubility of a certain component in the protein mixture.

3.1. pH control and isoelectric point precipitation

Proteins are generally less soluble at their isoelectric point.

3.2. Organic solvent separation method

The solubility of protein in different solvents is very different, ranging from basically insoluble (<10ug to="" extremely="" soluble="">300mg/ml). The concentration of the organic solvent that causes protein precipitation is different, so the concentration of the organic solvent can be controlled to separate the protein. Water-soluble non-ionic polymers such as polyethylene glycol can also cause protein precipitation.

3.3, temperature

Different proteins have different solubility and activity at different temperatures. Most proteins are relatively stable at low temperatures, so the separation operation is generally carried out at 0°C or lower.


The net charge of a protein depends on the sum of the positive and negative charges carried by the amino acid residues. For example, a neutral solution with a net negative charge is called an acidic protein.

4.1 Electrophoresis

It is not only an important means for separating protein mixtures and identifying protein purity, but also a very useful method for studying protein properties. The resolution of isoelectric focusing is very high, and the difference of pI can be separated by 0.02pH. The resolution of 2D-PAGE separation of proteins has been developed to 100,000 protein spots.

4.2. Ion exchange chromatography (IEX)

Change the salt ionic strength, pH, and (anion, cation) ion exchange packing in the protein mixture solution. Different proteins have different adsorption capacities for different ion exchange packings. Proteins are separated due to different adsorption capacities or non-adsorption.

The elution can be done by keeping the eluent composition constant, or by changing the salinity or pH of the eluent. The latter can be divided into segmented elution and gradient elution. Gradient elution generally has better effect and high resolution, especially for ion exchangers with small exchange capacity and sensitive to salt concentration. Gradient elution is often used. By controlling the volume of the eluent (compared to the column bed volume), salt concentration and pH, the sample components can be eluted separately from the ion exchange column.


5.1 Hydrophobic Chromatography (HIC)

Most of the hydrophobic amino acid residues are hidden inside the protein, but some are on the surface. The number and spatial distribution of the hydrophobic amino acid residues on the protein surface determine whether the protein has the ability to combine with the hydrophobic column packing to use it for separation. Because it is cheap and the purified protein has biological activity, it is a universal tool for separating and purifying protein. 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, so it is especially suitable for the mother liquor after precipitation and separation of concentrated ammonium sulfate solution and the target product after the precipitation is dissolved by salt. The solution is directly injected onto the column, and of course, the therapeutic protein extract of E. coli with 7 mol/L guanidine hydrochloride or 8 mol/L urea is also directly injected onto the column, and the refolding is carried out at the same time as the separation.

Affinity chromatography (AC)

Combines the characteristics of high efficiency and fast separation speed. Ligands can be enzyme substrates, inhibitors, cofactors, specific antibodies. After adsorption, the ionic strength and pH of the buffer can be changed, and it can be eluted. It can also be eluted with a higher concentration of the same ligand solution or a ligand solution with stronger affinity. According to the different ligands, it can be divided into:

6.1 Metal chelating medium

The transition metal ions Cu2+, Zn2+ and Ni2+ are bonded to the stationary phase in the form of imine complexes. Because these metal ions form coordination bonds with tryptophan, histidine and cysteine, The imine metal-protein chelate is formed, and the protein containing these amino acids is adsorbed by the stationary phase of this metal chelate affinity chromatography. The stability of the chelate is controlled by the dissociation constant of a single histidine and cysteine, which is also affected by the pH and temperature of the mobile phase. The controlled conditions can separate different proteins from each other.

6.2. Small ligand affinity medium

Ligands are arginine, benzamide, calmodulin, gelatin, heparin, lysine and so on.

6.3. Antibody affinity medium

That is, immunoaffinity chromatography, the ligands include recombinant protein A and recombinant protein G, but protein A is more specific than protein G, and protein G can bind more IgG from different sources.

6.4, pigment affinity medium

The effect of dye chromatography mainly depends on the affinity between the dye ligand and the enzyme, but also the type of elution buffer, ionic strength, pH value and the purity of the sample to be separated. There are two kinds of ligands: Cibacron Blue and Procion Red. Under certain conditions, the immobilized dye can act as a cation exchanger. In order to avoid this phenomenon, it is best to operate when the ionic strength is less than 0.1 and the pH is greater than 7.

6.5, lectin affinity medium

Ligands include concanavalin, lentil lectin and malt lectin. The solid-phase lectin can react reversibly with several carbohydrate residues and is suitable for the purification of polysaccharides and glycoproteins.


7.1, thermal stability

Most proteins will unfold or precipitate when heated to 95°C. Using this property, a protein that retains its soluble activity after such heating can be easily separated from most other cellular proteins.

7.2. Stability of proteolysis

Treat the supernatant with protease to digest the contaminated protein and leave the protein resistant to proteolysis.

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