With the development of biotechnology, more and more proteins and polypeptide chains are designed, manufactured and produced through the application of biotechnology such as genetic engineering, protein engineering and fermentation engineering.
In order to study and apply a certain protein, the target protein must first be separated from mixed protein molecules and non-some protein molecules.
Therefore, the separation and purification of recombinant proteins is widely used in biochemical research applications and is an important operational technology.
General steps for protein separation and purification
Material pretreatment and cell disruption
When separating and purifying a certain protein, it needs to be pretreated according to the way the protein is expressed. For intracellularly expressed proteins, the protein must first be released from tissues or cells and kept in its original natural state without losing activity.
Therefore, appropriate methods should be used to break up tissues and cells. Commonly used methods of disrupting tissue cells are as follows:
1.mortar and pestle
Grinding and breaking the wall is usually done by placing the plant tissue sample in frozen liquid nitrogen and grinding it with a tissue grinder. When tissue material is destroyed, metabolites can be extracted by adding solvents.
2.bead milling
The bead milling method refers to the method of adding glass beads or magnetic beads to an equal amount of cell suspension and a vortex mixer to lyse cells by the action of solid shear.
The mechanical shear force of this cell lysis method is gentle enough to maintain the integrity of organelles, has a high cell disruption rate, can be used on a large scale, and is suitable for various cell disruptions. The disadvantages are: macromolecular content is easily inactivated, and slurry separation is difficult.
3.Ultrasonic Breaker
Ultrasonic homogenizers work by inducing vibrations in a titanium probe immersed in a cell solution. A process called cavitation occurs in which tiny bubbles form and explode, creating localized shock waves and disrupting cell walls through pressure changes.
This method works well for plant and fungal cells, but has a drawback: it is noisy, so it must be done in an extra room.
4.homogenizer crushing method
The homogenizer uses shear forces on the cells similar to the bead method. Homogenization can be done by squeezing the cells into tubes slightly smaller than they are, thus shearing off the outer layer (French press) or by using the rotating blades in a blender (rotor-stator processor).
5.freeze crushing
Freeze-thaw cycles work by forming ice crystals and expanding cells upon thawing, eventually leading to rupture. For algae and soft plant material. The disadvantage is that it is very time consuming.
6.high temperature crushing method
Heat and pressure can break bonds in cell walls and can also denature proteins. Although fast, if your application is subject to thermal damage to the rest of the battery, you’d better find another way.
7.enzyme
Naturally occurring enzymes can be used to remove cell walls, for example when isolating protoplasts, depending on the biological tissue sample you use, lysozymes such as cellulase, chitinase, lysozyme, mannose, glycanase, etc. can be used.
protein extraction
In the process of protein extraction, an appropriate solvent is usually selected to extract the protein. The selection of conditions such as the composition, pH, and ionic strength of the buffer solvent used in the extraction should be determined according to the properties of the protein to be prepared.
For the extraction of membrane proteins, surfactants are generally added to the extraction buffer to destroy the membrane structure and facilitate the separation of proteins from the membrane. During the extraction process, attention should be paid to the temperature and avoid vigorous stirring to prevent protein denaturation. Insoluble matter such as cell debris can be removed by centrifugation or filtration.
1.isoelectric point precipitation
Different proteins have different isoelectric points, and they can be separated from each other by isoelectric point precipitation.
2.Salting out method
Salting-out refers to the phenomenon that a neutral salt is added to an aqueous protein solution, and the protein precipitates out as the salt concentration increases. Salting out is a technique for separating proteins based on their differences in solubility.
Neutral salt is a strong electrolyte with high solubility. In the protein solution, on the one hand, it competes with the protein for water molecules and destroys the water film on the surface of the protein colloidal particles;
On the other hand, it neutralizes the charge on the protein particles in a large amount, so that the protein particles in the water accumulate and precipitate out. Commonly used neutral salts include ammonium sulfate, sodium chloride, sodium sulfate, etc., but ammonium sulfate is the most common.
The protein obtained by salting out is generally not inactivated, and can be re-dissolved under certain conditions. Therefore, this method of precipitating protein is widely used in the work of separation, concentration, storage and purification of protein.
3.organic solvent precipitation
Neutral organic solvents such as ethanol and acetone have a lower dielectric constant than water. It can reduce the solubility of most globular proteins in aqueous solution, and then precipitate out of solution, so it can be used to precipitate proteins.
In addition, organic solvents will destroy the hydration layer on the surface of the protein, causing protein molecules to become unstable and precipitate out. Since organic solvents can denature proteins, it is necessary to operate at low temperature and select an appropriate concentration of organic solvents when using this method.
Further separation and purification of samples
The protein obtained by isoelectric point precipitation and salting-out method generally contains other protein impurities, which must be further separated and purified to obtain a sample with a certain purity. The commonly used purification methods are: gel filtration chromatography, ion exchange cellulose chromatography, affinity chromatography, etc. Sometimes these methods need to be used in combination to obtain higher purity protein samples.
1.gel filtration chromatography
Gel filtration chromatography, also known as size exclusion chromatography or molecular sieve method, is mainly used to separate and purify proteins according to their size and shape, that is, their quality.
The filler in the chromatographic column is some inert porous network structure substances, mostly cross-linked polysaccharide substances, so that the substances in the protein mixture can be separated according to the molecular size, and the macromolecular substances are excluded from the outside and flow down. The path is short and flows out first, while small molecular substances can enter the interior of the chromatography packing, and the path is longer when flowing down, so they will flow out later.
2.Ion exchange chromatography
Ion exchange chromatography separation is a separation method based on the different isoelectric points of proteins, and when the proteins are under different pH conditions, the charges on the proteins are different.
In ion exchange chromatography, the matrix is composed of charged resin or cellulose. Those with a positive charge are called anion exchange resins; those with a negative charge are called cationic resins. Ion exchangers commonly used for protein separation include weak acid type carboxymethyl cellulose and weak base type diethylaminoethyl cellulose.
3.Hydrophobic Interaction Chromatography
Hydrophobic chromatography is also called chromatography under hydrophobic interaction. From the perspective of separation and purification mechanism, it also belongs to the category of adsorption chromatography.
Hydrophobic chromatography uses hydrophobic ligands coupled to the stationary phase carrier to reversibly combine with some hydrophobic molecules in the mobile phase for separation. Generally, there are hydrophobic and hydrophilic groups on the surface of proteins. Hydrophobic chromatography uses a certain part of the protein surface to be hydrophobic, and binds to a hydrophobic carrier at a high salt concentration.
During elution, the salt concentration is gradually reduced, and they are eluted and purified one by one due to their different hydrophobicities, which can be used to separate proteins that are difficult to purify by other methods.
4.Affinity chromatography
Affinity chromatography is a chromatographic method that exploits specific interactions between biomolecules to separate molecules.
Affinity chromatography links molecules with a certain binding ability to the substances to be separated on the gel filtration chromatography column, and their binding is reversible, and the two can be separated from each other when the mobile phase conditions are changed.
Affinity chromatography can be used to purify or concentrate a molecule from a mixture, or it can be used to remove or reduce the amount of a molecule in a mixture.