What are the methods for extracting protein?

As we all know, protein is indispensable for human life activities. Many foods contain protein, and a normal diet can supplement protein for the body. In fact, in the field of biochemical research, protein separation and extraction technology has a wide range of applications. It is not easy to extract protein. It requires many processes and professional operating techniques. Now there are the following methods to extract protein.

1. Ultracentrifugation

The principle of this method for separating and purifying antigens is to utilize the different sedimentation rates of each particle in the gradient solution so that particles with different sedimentation rates are in different density gradient layers to achieve the purpose of separation from each other. Commonly used density gradient media include sucrose, glycerol, CsCl, etc.

When separating and purifying antigens by ultracentrifugation or gradient density centrifugation, it is extremely difficult to separate a certain antigen component except for individual components. Therefore, it is only used to separate a few large molecular antigens, such as IgM, C1q, thyroglobulin, etc., and some lighter antigen substances such as apolipoprotein A, B, etc. Most medium and small molecular weight proteins are difficult to purify using this method.

2. Selective precipitation method

The principle is to use various precipitants or change certain conditions to induce the precipitation of protein antigen components based on the differences in the physical and chemical properties of each protein, thereby achieving the purpose of purification. The most commonly used method is salting out precipitation.

Principle of salting out method

The solubility of protein in aqueous solution depends on the number of water molecules around the surface ions of the protein molecules, that is, it is mainly determined by the degree to which the hydrophilic groups on the periphery of the protein molecules form a hydration film with water and the charge of the protein molecules. After adding neutral salt to the protein solution, the hydration layer around the protein molecules is weakened or even disappears because the affinity of neutral salt to water molecules is greater than that of protein. At the same time, when neutral salt is added to the protein solution, the ionic strength changes and the charge on the protein surface is largely neutralized, which further reduces the solubility of the protein and causes the protein molecules to aggregate and precipitate. Since various proteins have different solubility in different salt concentrations, salt solutions of different saturations precipitate different proteins, thereby separating them from other proteins. The most commonly used salt solution is ammonium sulfate at 33% to 50% saturation. The salting-out method is simple and convenient and can be used for crude extraction of protein antigens, extraction of immunoglobulin G, protein concentration, etc. The purity of antigens purified by the salting-out method is not high and is only suitable for the preliminary purification of antigens.

3. Gel chromatography

Gel chromatography uses molecular sieving to separate proteins. Gel is a substance with a three-dimensional porous network structure. After proper solution balance, it is loaded into the chromatography column. When a sample solution containing various molecules slowly flows through a gel chromatography column, macromolecular substances cannot easily enter the micropores of the gel particles and can only be distributed between the particles. Therefore, they move downward faster during elution and are eluted first. In addition to diffusing in the gaps between gel particles, small molecules can also enter the micropores of the gel particles, move downward more slowly during elution, and are subsequently eluted. Therefore, protein molecules are separated according to their molecular size.

4. Ion exchange chromatography

The principle of ion exchange chromatography is to use some cellulose or gel with ionic groups to adsorb and exchange protein antigens with opposite charges. Because various proteins have different isoelectric points and carry different amounts of charge, their abilities to bind to cellulose (or gel) vary. During gradient elution, the ionic strength of the mobile phase is gradually increased, so that the added ions compete with the proteins for the charge positions on the cellulose, thereby dissociating the adsorbed proteins from the ion exchanger.

The following ion exchangers are commonly used in ion exchange chromatography technology:

① Cellulose with ion exchange groups, such as carboxymethyl (CM) cellulose, DEAE-cellulose

② Cross-linked dextran, agarose and polyacrylamide with ion exchange groups

③Highly cross-linked resin synthesized by gel.

5. Affinity chromatography

Affinity chromatography is a chromatography technique designed to utilize the biospecificity of biomacromolecules, that is, the specific affinity between biomacromolecules. For example, there is a special affinity between antigens and antibodies, enzymes and enzyme inhibitors (or ligands), enzyme proteins and coenzymes, hormones and receptors, IgG and Staphylococcal protein A (SPA). For example, when purifying IgG, SPA can be adsorbed on an inert solid phase matrix (such as Spehrose 2B, 4B, 6B, etc.) and prepared into a chromatography column. When the sample flows through the chromatography column, the IgG to be separated can specifically bind to SPA, while the other components cannot bind to it. After the chromatography column is fully eluted, the ionic strength or pH value of the eluent is changed to dissociate the IgG from the SPA on the solid phase matrix, and the IgG to be purified can be obtained by collecting the eluent.