The separation and purification of proteins is widely used in biochemical research applications and is an important operating technique. Protein purification from other proteins and non-protein molecules is a very complex process, and there are also some pitfalls, let's take a look at the path that brothers and sisters have traveled for you.

Protein purification principles

Protein purification should use the internal similarity and differences between different proteins, use the similarity between various proteins to remove the contamination of non-protein substances, and use the differences of each protein to purify the target protein from other proteins. Each protein has differences in size, shape, charge, hydrophobicity, solubility, and biological activity, which can be used to extract proteins from mixtures such as E. coli lysate to obtain recombinant proteins.

The purification of proteins is roughly divided into two stages: coarse separation stage and fine purification stage. The general method used for protein purification is the resin method. Due to the large volume and complex components of the sample, the resin used requires high capacity, high flow rate, large particle size, wide particle size distribution, and can quickly separate the protein from the contaminants, and if necessary, corresponding protective agents (such as protease inhibitors) can be added to prevent the target protein from being degraded.

The fine purification stage requires higher resolution, which is to distinguish the target protein from those proteins with similar molecular weight and physical and chemical properties, and to use smaller resin particles to improve resolution. Selectivity refers to the specificity of the binding of the resin to the target protein, while column effect refers to the ability of each protein component to be eluted from the resin one by one. Only good selectivity, the elution peak is too wide, and the protein is still not effectively separated.

Procedure

The general procedure for separating and purifying a specific protein can be divided into three steps: pretreatment, coarse fractionation, and subdivision.

Pre-treatment

To isolate and purify a certain protein, the protein must first be released from the original tissue or cell in a dissolved state and maintain its original natural state without losing biological activity. To this end, animal material should first remove connective tissue and adipose tissue, seed material should be shelled or even degreased to avoid tannins and other substances, and oil seeds should be degreased with organic solvents with low boiling points such as ether. Then, depending on the situation, choose the appropriate method to break up the tissue and cells. Animal tissues and cells can be crushed with an electric masher or homogenizer or treated with ultrasound. Plant tissues and cells generally need to be ground with quartz sand or glass powder with appropriate extracts or treated with cellulase due to their cell walls composed of cellulose, hemicellulose, and pectin. The fragmentation of bacterial cells is more troublesome because the skeleton of the entire bacterial cell wall is actually a peptidoglycan sac-like macromolecule connected by covalent bonds, which is very tough. Common methods for breaking bacterial cell walls include ultrasonic crushing, sand grinding, high-pressure extrusion, or lysozyme treatment. After the tissue and cells are broken up, select the appropriate buffer to extract the desired protein. Insoluble substances such as cell debris are removed by centrifugation or filtration.

If the desired protein is mainly concentrated in a certain cell component, such as the nucleus, chromosomes, ribosomes, or soluble cytoplasm, they can be separated by differential centrifugation and the cell component can be collected as material for further purification. If the protein is bound to the cell membrane or membranous organelles, the membrane structure must be depolymerized by ultrasound or detergent and then extracted with appropriate media.

Coarse separation

When the protein extract (sometimes mixed with nucleic acids, polysaccharides, etc.) is obtained, an appropriate method is used to separate the desired protein from other heteroproteins. Generally, this step of separation uses methods such as salt precipitation, isoelectric point precipitation and fractional separation of organic solvents. These methods are characterized by their simplicity and high throughput, removing large amounts of impurities and concentrating protein solutions. Some protein extraction liquids are large and not suitable for concentration by precipitation or salting, so ultrafiltration, gel filtration, freeze vacuum drying or other methods can be used for concentration.

Fine separation

After coarse separation, the sample is generally small in size, and most of the heteroproteins have been removed. For further purification, chromatography methods are generally used, including gel filtration, ion exchange chromatography, adsorption chromatography, and affinity chromatography. If necessary, electrophoresis methods can also be selected, including zone charge electrophoresis, isoelectric point focusing, etc. as the final purification step. The methods used for fractional separation are generally small in scale but have high resolution.

Crystallization is the final step in protein separation and purification. Although the crystallization process does not guarantee that proteins are uniform, crystallization can only be formed when a protein has a numerical advantage in solution. The crystallization process itself is accompanied by a certain degree of purification, and recrystallization removes a small amount of the entrapped protein. Since denatured proteins are never found during crystallization, the crystallization of proteins is not only a sign of purity, but also a strong indicator that the product is in its natural state.

Ion chromatography

When the separated protein solution flows through the ion exchange chromatography column, the protein with the opposite charge to the ion exchanger is adsorbed on the ion exchanger, and then the adsorbed protein is eluted by changing the pH.

Organic solvent extraction

The principle of protein purification organic solvent extraction is that organic solvents that are miscible with water (such as methanol and ethanol) can significantly reduce the solubility of some proteins in water. Moreover, at a certain temperature, pH and ionic strength, the concentration of organic solvents causing protein precipitation is different, so controlling the concentration of organic solvents can separate and purify proteins.

For example, the slow addition of ethanol (-25°C) to a pre-chilled culture medium at 4°C under magnetic stirring in an ice bath can precipitate the ice and nucleus proteins, thereby purifying the ice proteins. Since at room temperature, organic solvents not only cause precipitation of proteins, but also accompany denaturation. Therefore, the problem of protein denaturation can be largely solved by cooling the organic solvent and then adding the organic solvent under constant stirring to prevent the local concentration from being too high. For some proteins that are strongly bound to lipids or have more polar side chains in the molecule and are insoluble in water, they can be extracted with organic solvents such as ethanol, acetone and butanol, which have a certain degree of hydrophilicity and strong lipophilicity, and are ideal extraction solutions. The cold ethanol separation method was first proposed by Cohn in 1949 for the preparation of gamma globulin. The cold ethanol method is also a method recommended by WHO regulations and Chinese biological products regulations, which not only has high resolution, good purification effect, can separate multiple components at the same time, but also has the effects of antibacterial, scavenging and virus killing.

Guide to avoiding pits

Contamination of nucleic acid impurities such as DNA/RNA in samples is difficult to remove?

a. Extend the ultrasound time to reduce viscosity. Nucleases such as Benzonase can also be used to digest DNA and RNA at the same time. If the source is E. coli lysate, which contains a large amount of DNA, a large amount of DNA can be pre-removed by methods such as streptomycin precipitation. b. Removal by chromatography: Nucleic acids can also be effectively removed due to their negative charge, which binds on the anion column or flows through the cationic column.

Protein is old and not hanging up?

a. Improper ultrasound power: too much makes the protein carbonized, and too small protein is not released. It is recommended to adjust the ultrasound power or add lysozyme before ultrasound to increase cell fragmentation. b. Unsuitable buffer conditions: The concentration of metal ion chelating agents such as EDTA and citric acid in the buffer should not be too high, and the pH can also be appropriately raised. c. Inadequate exposure of His tags: Purification by adding an appropriate amount of urea or a denaturant such as guanidine hydrochloride to the protein or changing the length or position of His at the upstream molecular level exposes it to the protein surface. d. Inadequate exposure of His tags: Purification by adding an appropriate amount of urea or a denaturant such as guanidine hydrochloride to the protein or changing the length or position of the upstream molecule level exposes it to the protein surface. e. Column Overload: Replace large volume columns or multi-column series.

Cannot elute the protein after hanging the column?

a. Elution conditions are too mild: increase the concentration of imidazole in the buffer or appropriately reduce the pH of the buffer. b. Non-specific hydrophobic or other interactions: adding nonionic detergents or increasing the concentration of NaCl. c. Protein precipitation: appropriately reduce the loading amount or protein concentration, and use imidazole for linear elution; Additives are used to either change the concentration of NaCl or elute under denaturing conditions.

How many impurities are there after protein elution?

a. Protease degradation of some target proteins: adding protease inhibitors to improve b. High affinity between heteroproteins and elution columns: optimizing imidazole concentrations or improving pH, NaCl concentrations c. Interaction between impurity proteins and target proteins:Add a detergent or reducing agent before ultrasound to reduce non-specific effects

With the deepening of molecular biology, structural biology, genomics and other research, people realize that it is not enough to rely solely on genome sequence analysis to try to elucidate the phenomena and essence of life activities. Only by studying the sum of all proteins from the perspective of proteomics can we more scientifically grasp the phenomena and laws of activity of life, and more fully reveal the essence of life.