Biotechnology is developing. More and more proteins and polypeptide chains are designed, engineered, and produced. They use biotechnological approaches. These include genetic engineering, protein engineering, and fermentation engineering.
You want to study and apply a specific protein. You must first separate the target protein from a mixture. The mixture has other proteins and non-protein molecules.
So, the separation and purification of recombinant proteins are widely used in biochemical research and applications. They are a critical operational technology.
1. General Procedures for Protein Separation and Purification
Material Pretreatment and Cell Disruption
You isolate and purify a specific protein. You need pretreatment. It depends on the protein expression system. The ptn is expressed inside cells. The first step is to release the ptn from tissues or cells. You must keep its native conformation and biological activity.
So, you need to use appropriate methods. These methods disrupt tissues and cells. Common cell and tissue disruption methods are as follows:
Mortar and Pestle
Grinding-based cell disruption usually involves plant tissue samples. You put them in frozen liquid nitrogen. You grind them with a tissue grinder. The tissue material is disrupted. You can add solvents. They extract metabolites.
Bead Milling
Bead milling is a method. You add glass beads or magnetic beads. You add them to an equal volume of cell suspension. This is done in a vortex mixer. Cell lysis is achieved through solid shear forces.
This mechanical cell disruption method uses shear forces. The forces are sufficiently mild. They preserve organelle integrity. It has high cell disruption efficiency. It is suitable for large-scale applications. It works for various cell types. It has disadvantages. Macromolecules are likely to become inactive. The resulting slurry is hard to separate.
Ultrasonic Cell Disruptor
An ultrasonic homogenizer works like this. A titanium probe is immersed in the cell solution. It induces vibrations. This process triggers cavitation. Microbubbles form and implode. Localized shock waves are generated. They disrupt cell walls through pressure changes.
This method is particularly suitable for plant and fungal cells. It has a drawback. It produces high levels of noise. You need to operate it in a separate room.
Homogenizer Disruption
Homogenizers apply shear forces. They are similar to those in bead milling. They act on cells. Homogenization can be done in two ways. One way is forcing cells through a tube. The tube is slightly smaller than their diameter. This shears the outer layer (French press). The other way is using rotating blades in a blender (rotor-stator processor).
Freeze-Thaw Disruption
Freeze-thaw cycles work like this. Ice crystals form. Cells expand during thawing. This leads to cell rupture. This method is used for algae and soft plant materials. It has a main disadvantage. It takes a lot of time.
High-Temperature Disruption
Temperatures and pressures are elevated. They break the bonds within cell walls. They also cause protein denaturation. This method is fast. Your application is sensitive to thermal damage. The damage affects other cellular components. You should use alternative methods.
Enzymatic Lysis
Naturally occurring enzymes can be used. They remove cell walls. For example, during protoplast isolation. You use biological tissue samples. You can employ lytic enzymes. These include cellulase, chitinase, lysozyme, mannanase, and glycanase.
2. Protein Extraction
In the protein extraction process, you usually select an appropriate solvent. It extracts the target ptn. The extraction buffer has composition, pH, and ionic strength. You determine them based on the properties of the target it.
For example, you extract membrane proteins. You generally add surfactants to the extraction buffer. They disrupt membrane structures. They help separate proteins from membranes. During the extraction process, you need to pay attention to temperature control. You should avoid vigorous stirring. This prevents ptn denaturation. Insoluble substances like cell debris can be removed. You use centrifugation or filtration.
Isoelectric Point Precipitation
Different proteins have distinct isoelectric points. This allows their separation. You use isoelectric point precipitation.
Salting-Out
Salting-out is a phenomenon. It precipitation occurs in an aqueous ptn solution. The concentration of neutral salts increases. It is a separation technology. It is based on differences in ptn solubility.
Neutral salts are strong electrolytes. They have high solubility. In ptn solutions, they compete with proteins for water molecules. They disrupt the hydration shell on the surface of ptn colloidal particles. At the same time, they neutralize a large number of charges on ptn particles. This causes the aggregation and precipitation of ptn particles in the aqueous solution. Common neutral salts include ammonium sulfate, sodium chloride, and sodium sulfate. Ammonium sulfate is the most widely used.
Proteins obtained via salting-out generally retain their biological activity. They can be redissolved under specific conditions. So, this ptn precipitation method is extensively applied. It is used in ptn separation, concentration, storage, and purification.
Organic Solvent Precipitation
Neutral organic solvents like ethanol and acetone have lower dielectric constants than water. They can reduce the solubility of most globular proteins in aqueous solutions. This leads to their precipitation. So, they can be used for ptn precipitation.
In addition, organic solvents disrupt the hydration layer on the ptn surface. They destabilize ptn molecules.
This causes them to precipitate. Organic solvents can induce ptn denaturation. So, this method requires operation at low temperatures. You need to carefully select appropriate organic solvent concentrations.
3.Further Separation and Purification of Samples
Proteins obtained through isoelectric point precipitation and salting-out typically contain other ptn impurities. You need further purification. You want to get samples of a certain purity. Common purification methods include gel filtration chromatography, ion-exchange cellulose chromatography, and affinity chromatography. In some cases, you need to combine these methods. You can get high-purity ptn samples.
Gel Filtration Chromatography
Gel filtration chromatography is also called exclusion chromatography or molecular sieve chromatography. It mainly separates and purifies proteins. It is based on their size and shape, or their molecular weight.
The packing material in the chromatography column consists of inert, porous network-structured substances. Most are cross-linked polysaccharides. This allows the separation of components in ptn mixtures. It is based on molecular size. Large molecules are excluded from the interior of the packing material. They travel a shorter path. They elute first. Small molecules can penetrate the packing material. They travel a longer path. They elute later.
Ion-Exchange Chromatography
Ion-exchange chromatography separates proteins. It is based on differences in their isoelectric points. Under different pH conditions, proteins carry different net charges. This is the basis for their separation.
In ion-exchange chromatography, the matrix consists of charged resins or celluloses. Resins with positive charges are called anion-exchange resins. Resins with negative charges are called cation-exchange resins. Common ion-exchange agents used for it separation include weakly acidic carboxymethyl cellulose (CMC) and weakly basic diethylaminoethyl cellulose (DEAE-cellulose).
Hydrophobic Interaction Chromatography (HIC)
Hydrophobic interaction chromatography is also called hydrophobic chromatography. It is classified as a type of adsorption chromatography. It is based on its separation and purification mechanism.
HIC uses reversible interactions. They are between hydrophobic ligands. The ligands are coupled to the stationary phase carrier. They interact with hydrophobic molecules in the mobile phase. This achieves separation. It surfaces generally have both hydrophobic and hydrophilic regions. HIC leverages the hydrophobic regions on ptn surfaces. They bind to hydrophobic carriers at high salt concentrations.
During elution, the salt concentration is gradually reduced. Proteins are eluted sequentially. It is based on their hydrophobicity. This achieves purification. This method can be used to separate proteins. They are difficult to purify using other techniques.
Affinity Chromatography
Affinity chromatography is a chromatographic method. It separates molecules. It uses specific biological interactions between them.
In affinity chromatography, molecules have specific binding affinity to the target substance. They are immobilized on a gel filtration chromatography column. The binding between the ligand and the target molecule is reversible. You can separate them when the mobile phase conditions are altered.
Affinity chromatography can be used to purify or concentrate a specific molecule from a mixture. It can also be used to remove or reduce the content of a specific molecule in a mixture.
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