Protein Isolation and Purification Techniques

Protein purification is crucial for protein identification, function, structure and interaction studies. Protein separation and purification is a method of separating and purifying the desired protein from the mixture using downstream technology of biological engineering, and is the core technology in the contemporary biological industry.

The method of protein purification is mainly to use the similarities and differences between different proteins. According to the similarities between proteins, non-protein substances can be removed, and then the target protein is separated according to the differences of the proteins. Separation and purification of proteins can be divided into various methods according to the nature of the protein itself.

Based on protein and structure and function, understanding the life scene from the molecular level has become the main direction of the development of modern biology. To study proteins, we must first obtain highly purified and biologically active target substances. The preparation of protein touches on various aspects of physics, chemistry and biology, but the basic principles are nothing more than two aspects. One is to use the difference in the distribution ratio of several components in the hybrid, and divide them into two or several phases that can be separated by mechanical methods, such as salting out, organic solvent isolation, chromatography and crystallization, etc.; The mixture is placed in a single phase, and after the action of the physical force field, the components are distributed in the same area to achieve separate goals, such as electrophoresis, ultracentrifugation, and ultrafiltration. In the application of all these methods, care must be taken to preserve the integrity of biological macromolecules, to avoid the acid, alkali, high temperature, and strong mechanical action resulting in the loss of biological activity of the substance. The preparation of protein is usually divided into the following four stages: selection of materials and pre-solution, cell disruption and separation of organelles,  isolation and purification, concentration, single harmony retention.

Microorganisms, plants and animals can all be used as raw materials for the preparation of proteins. The materials selected are mainly judged according to the experimental objectives. For microorganisms, attention should be paid to its growth period. In the logarithmic growth period of microorganisms, the content of enzymes and nucleic acids is high, and high yields can be obtained. There are two cases when microorganisms are used as materials:

(1) Metabolites and extracellular enzymes secreted into the culture medium by microbial cells must be used;

(2) Use the biochemical substances contained in the bacteria, such as proteins, nucleic acids and intracellular enzymes. The plant material must be dehulled, degreased, and pay attention to the different plant varieties and growth and development. The amount of biological macromolecules contained in it has changed greatly, and it is closely related to seasonality. For animal tissues, organ tissues with abundant effective ingredients must be selected as raw materials, and minced and degreased first. In addition, the pre-resolved materials should be kept frozen if the experiment is not carried out immediately, and fresh materials should be selected for the biodegradable macromolecules.

Separate Purification of Proteins

1. Isolation of proteins (including enzymes)

Most proteins are soluble in water, dilute salts, dilute acids or alkaline solutions, and a few proteins linked to lipids are soluble in organic solvents such as ethanol, acetone, butanol, etc. , You can take different solvents to extract and purify proteins and enzymes separately.

(1) Aqueous solution  isolation method

The aqueous solution of dilute salt and buffer system has good protein consolidation and high solubility. It is the most commonly used solvent for protein  isolation. The dosage is usually 1-5 times the volume of the raw material. It needs uniform stirring during  isolation to facilitate protein dissolution. The  isolation temperature depends on the nature of the active ingredients. On the one hand, the solubility of most proteins increases with increasing temperature. Therefore, high temperature facilitates dissolution and shortens the  isolation time. But on the other hand, the increase in temperature will denature and inactivate the protein. Therefore, when extracting proteins and enzymes based on this point, low temperature (below 5 degrees) is usually adopted. In order to avoid degradation during protein  isolation, proteolytic enzyme inhibitors (such as diisopropylfluorophosphate, iodoacetic acid, etc.) can be added.

The following focuses on the selection of pH and salt concentration of the extract.

1. pH value protein, enzyme is an ampholyte with isoelectric point, and the pH value of the  isolation solution should be selected in the pH area deviating from the isoelectric point on both sides. When extracting with dilute acid or alkali, avoid the change of protein dissociable group caused by overacid or overbase, which leads to irreversible change of protein conformation. Generally speaking, alkaline protein is extracted with acidic extract, For acidic proteins, use a slightly alkaline extract.

2. Salt concentration

Dilute concentration can improve the dissolution of protein, called salt dissolution. At the same time, the dilute salt solution has the advantage of protecting the protein from denaturation due to the partial connection of the salt ions and the protein. Therefore, a small amount of neutral salt such as NaCl is added to the extract, usually 0.15 mol. The concentration is suitable. The buffer solution is usually 0.02-0.05 M phosphate and carbonate isotonic saline solution.

(2) Organic solvent  isolation method

Some proteins and enzymes that are more strongly linked to lipids or have more non-polar side chains in the molecule are not soluble in water, dilute salt solutions, dilute acids or dilute bases. Organic solvents such as ethanol, acetone, and butanol can be used. It has certain hydrophilicity and strong lipophilicity, which is the ideal extract of lipoprotein. But it must be operated at low temperature. The butanol  isolation method is particularly advantageous for extracting some proteins and enzymes that are closely linked to lipids. First, butanol has strong lipophilicity, especially the ability to dissolve phospholipids; second, butanol has both hydrophilicity and solubility in the field of solubility. (Degree is 10%, 40 degrees is 6.6%) will not cause degeneration and inactivation of the enzyme. In addition, the butanol  isolation method has a wide range of pH and temperature selection, and is also suitable for animals, plants and microbial materials.

2. Separate purification of protein

There are many methods for purifying proteins separately, mainly:

(1) Separate methods according to different protein solubility

1. Salting out of protein

Neutral salt has obvious influence on the solubility of protein. Generally, the solubility of protein increases with the increase of salt concentration at low salt concentration. This is called salt dissolution; when the salt concentration continues to increase, the solubility of protein decreases to different degrees and successively The precipitation is called salting out. A large amount of salt is added to the protein solution. The high concentration of salt ions (such as SO4 and NH4 of ammonium sulfate) has a strong hydration power, which can grab the hydration layer of protein molecules, so that “Water loss”, so the protein colloid coagulates and deposits. The effect is better if the pH of the solution is at the isoelectric point of the protein during salting out. Because the particle size and hydrophilicity of various protein molecules are different, the salt concentration required for salting out is also different. Therefore, adjusting the neutral salt concentration in the mixed protein solution can cause various proteins to be deposited in stages.

The factors affecting salting out are:

(1) Temperature: In addition to temperature-sensitive proteins operating at low temperatures (4 degrees), they can usually be performed at room temperature. Usually the protein solubility decreases at low temperatures. However, some proteins (such as hemoglobin, myoglobin, albumin) are more soluble at higher temperatures (25 degrees) than at 0 degrees, and are more likely to salt out.

(2) PH value: Most proteins have the lowest solubility in concentrated salt solution at the isoelectric point.

(3) Protein concentration: When the protein concentration is high, the protein to be separated often accumulates together with the rest of the protein (together the scene). Therefore, before the salting out, the serum should be diluted with the same amount of normal saline to make the protein content 2.5-3.0%.

Commonly used neutral salts for protein salting out are mainly ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride, sodium phosphate, etc.

Among them, the most used ammonium sulfate has the advantages of small temperature coefficient and high solubility (saturated solution at 25 degrees is 4.1M, which is 767 grams/liter; at 0 degrees, the saturated solubility is 3.9M, or 676 grams/liter) In this solubility field, many proteins and enzymes can be salted out; in addition, the ammonium sulfate staged salting out effect is also better than the rest of the salt, which is not easy to cause protein denaturation. The pH of the ammonium sulfate solution is usually between 4.5-5.5. When the remaining pH value is used for salting out, it needs to be adjusted with sulfuric acid or ammonia.

After the protein is deposited by salting out, the salt in the protein needs to be removed. The common method is dialysis, that is, the protein solution is put into the Xiu analysis bag (usually cellophane), and the buffer is used for dialysis, and it is constantly changed. Since the buffer solution requires a longer time for dialysis, it is best to perform it at a low temperature. In addition, the G-25 or G-50 Glucose column can also be used to remove salt, and the time used is relatively short.

2. Isoelectric point deposition method

When the protein is static, the electrostatic repulsion between the particles is the smallest, so the solubility is also the smallest, and the isoelectric points of various proteins are different. The pH of a solution can be adjusted to reach the isoelectric point of a protein to deposit it, but this method is very Less used alone, can be used in conjunction with salting out method.

3. Low temperature organic solvent deposition method

The use of water-miscible organic solvents, methanol, ethanol or acetone, can reduce the solubility of most proteins and precipitate out. This method has higher discrimination than salting out, but the protein is more volatile and should be carried out at low temperatures.

(2) Separate methods based on the difference in protein molecular size

1. Dialysis and ultrafiltration

Dialysis uses semi-permeable membranes to separate proteins of different molecular sizes.

The ultrafiltration method uses high pressure or centrifugal force to force water and other small solute molecules to pass through the semi-permeable membrane, while the protein is left on the membrane. A lubricating membrane with different pore sizes can be selected to retain proteins of different molecular weights.

2. Gel filtration method

Also known as size exclusion chromatography or molecular sieve chromatography, this is one of the most effective methods for protein mixtures based on molecular size. The most commonly used packing materials in columns are glucose gel (Sephadexged) and agarose gel (agarosegel).

(3) According to the charged properties of the protein, the charged properties and the number of charges of the protein in different pH environments are different, and they can be separated.

1. Electrophoresis

Under the same pH condition, various proteins can be separated due to different mobility in the electric field due to different molecular weight and charge quantity. It is worth noting that isoelectric focusing electrophoresis, which uses a kind of ampholyte as a carrier, the ampholyte forms a pH gradient that gradually increases from the positive electrode to the negative electrode during electrophoresis. When a certain charged protein swims in it, it reaches their respective The pH position of the electrical point is stagnant, and this method can be used to analyze and prepare various proteins.

2. Ion exchange chromatography

Ion exchangers include cation exchangers (such as: carboxymethyl cellulose; CM-cellulose) and anion exchangers (diethylaminoethyl cellulose), when separate protein solutions flow 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 or ionic strength.

(4) Separate methods based on the magical nature of ligands—affinity chromatography Affinity chromatography (aflinitychromatography)

is an extremely effective method for separating proteins. It often requires only one step to resolve a certain type of protein to be purified Proteins are separated from very complex protein mixtures, and the purity is very high. This method is based on the fact that certain proteins can be magically linked to another molecule called a ligand (Ligand) rather than covalently. Its basic principle: Proteins exist in a complex mixture in tissues or cells. Each type of cell contains thousands of different proteins, so the separation, purification and characterization of proteins It is an important part of biochemistry. There is no single method or a set of ready-made methods that can extract any kind of protein from the miscellaneous mixed protein, so it is often used in several ways.

Cell fragmentation

1. High speed tissue smashing: mix the material into a thin paste-like liquid, place it in the cylinder for about 1/3 volume, close the cylinder cover tightly, dial the governor to the slowest place first, and after the switch is turned on, gradually accelerate to all Need speed. This method applies to animal visceral tissues, plant fleshy seeds, etc.

2. Glass homogenizer homogenization: first put the shredded tissue in the tube, then put it into the grinding rod to grind back and forth, and move it up and down to grind the cell. The degree of cell breakage is higher than that of the high-speed tissue masher. High, suitable for small amounts and animal organs.

3. Ultrasonic solution: use a certain power of ultrasonic to solve the cell suspension and make the cells vibrate and rupture rapidly. This method is mostly suitable for microbial materials. E. coli is used to prepare various enzymes. The concentration of 50-100 mg bacteria/ml is often used. Solve for 10-15 minutes at a frequency of 1KG to 10KG. The disadvantage of this method is that a large amount of heat will be generated during the solution process, and the corresponding cooling method should be adopted. Sensitive to ultrasound and nucleic acids should be used with caution.

4. Repeated freeze-thaw method: The cells are frozen below -20 degrees Celsius, thawed at room temperature, and repeated several times, because the formation of ice particles in the cells and the increase in the salt concentration of the remaining cell fluid cause swelling and break the cell structure.

5. Chemical solution: Some animal cells, such as tumor cells, can be damaged by cell membranes such as sodium dodecyl sulfonate (SDS) and sodium deoxycholate. The bacterial cell wall is thick, and lysozyme can be used to solve the effect better.

No matter which method is used to break up tissue cells, it will cause intracellular protein or nucleic acid hydrolase to be released into the solution, causing macromolecules to biodegrade, resulting in a decrease in the quality of natural substances. The addition of diisopropylfluorophosphate (DFP) can inhibit or Slow down autolysis; adding iodoacetic acid can inhibit the activity of those proteolytic enzymes that require a sparse base in the active center, and adding benzenesulfonyl fluoride (PMSF) can also eliminate the proteolytic activity, but not all, but also through Select pH, temperature or ionic strength, etc., so that these conditions are suitable for the  isolation of the target substance.

Concentration, monotony and retention

1. Concentration of Samples

Biomacromolecules become very dilute due to column purification during the preparation process. In order to retain and identify the target, they often need to be concentrated. Commonly used concentration methods:

1. Vacuum concentration, evaporation and concentration

After decreasing the liquid surface pressure, the boiling point of the liquid is lowered. The higher the vacuum degree of decompression, the lower the boiling point of the liquid and the faster the evaporation. This method is suitable for the concentration of some heat-resistant biological macromolecules.

2. Air flow evaporation concentration

The flow of air can accelerate the evaporation of the liquid, spread into a thin layer of solution, and the surface continues to pass through the air flow; or put the biological macromolecule solution into the dialysis bag and install it in the cold room, use the electric fan to direct the air to make the solvent through the membrane It does not evaporate, but reaches the concentration target. This method is slow in concentration and is not suitable for the concentration of a large number of solutions.

3. Freezing method

Biomacromolecules form ice at low temperatures. Salts and biomacromolecules do not enter the ice but remain in the liquid phase. During operation, the solution to be concentrated is first cooled to become a solid, and then slowly melted, using solvents and solutes The difference between the melting point and the intermediate point achieves the goal of removing most of the solvent. For example, when the salt solution of protein and enzyme is concentrated by this method, pure ice crystals without protein and enzyme float on the liquid surface, and protein and enzyme are concentrated in the lower solution, and the upper ice block is removed to obtain the concentration of protein and enzyme. liquid.

4. Absorption method

After being absorbed, the solution molecules in the solution are directly removed and concentrated. The absorbent used must not react chemically with the solution and does not adsorb biological macromolecules, so it is easy to separate from the solution. Commonly used absorbents include polyethylene glycol, polyvinylpyrrolidone, sucrose and gel, etc. When using polyethylene glycol absorbents, first put the biomacromolecule solution in a bag of semi-permeable membrane, and add polyethylene glycol The alcohol cover is placed at 4 degrees. The solvent in the bag oozes and is quickly absorbed by the polyethylene glycol. After the polyethylene glycol is saturated with water, it must be replaced with a new one until the required volume is reached.

5. Ultrafiltration method

The ultrafiltration method uses a special membrane to selectively filter various solute molecules in the solution. When no liquid passes through the membrane under a certain pressure (nitrogen pressure or vacuum pump pressure), the solvent and small molecules permeate, large molecules Wall retention, this is a new method developed in recent years. It is most suitable for the concentration or desalination of biological macromolecules, especially proteins and enzymes. It has low cost, convenient operation, soft conditions, and can better maintain the activity of biological macromolecules. , High recovery rate and other advantages. The key to using ultrafiltration is the choice of membrane. Different types and specifications of membrane, water flow rate, molecular weight cut-off value (that is, the minimum molecular weight value of molecules that can be retained by the membrane) and other parameters are different, and must be selected according to the needs of the work. In addition, the situation of the ultrafiltration device, solute components and properties, solution concentration, etc. all have a certain impact on the ultrafiltration effect.

A hollow fiber tube is made with the above ultrafiltration membrane, and many such tubes are gathered into a bundle. Both ends of the tube are connected with a buffer solution of low ionic strength, so that the buffer solution continuously flows in the tube. Thereafter, the fiber tube is immersed in the protein solution to be dialyzed. When the buffer flows through the fiber tube, small molecules can easily diffuse through the membrane and large molecules cannot. This is the fiber filtration method, because the dialysis area increases, so the dialysis time is shortened by 10 times.

2. Monotonous

Products prepared from biological macromolecules are often monotonous to avoid deterioration and easy to retain. The most commonly used method is freezing and harmonious vacuum monotony. Vacuum monotony is suitable for the single harmonious preservation of substances that are not resistant to high temperature and easy to oxidize. All devices include monotoners, condensers and vacuum monotonic principles, while increasing the temperature factor. At the same pressure, the water vapor pressure drops as the temperature drops, so at low temperatures and pressures, ice easily sublimates into gas. During operation, the liquid to be monotonous is usually frozen below the freezing point to make it solid, and then the solvent is turned into gas and removed at low temperature and low pressure. The dried product of this method has the advantages of fluffiness, good solubility, and maintenance of natural structure. It is suitable for the monotonous retention of various biological macromolecules.

3. Storage

The consolidation of biological macromolecules has a great relationship with the retention method. Monotonous products are usually more consolidated, and their activity can be changed without obvious changes in days or even years at low temperatures. It is easy to plead for storage. Simply place the monotonous sample in a monotoner (with monotonic solution inside) to seal and maintain 0- A 4 degree refrigerator is sufficient. Pay attention to the following points during liquid storage.

1. The sample should not be too dilute. It must be concentrated to a certain concentration and encapsulated for storage. If the sample is too dilute, it will easily denature biological macromolecules.

2. It is usually necessary to add preservatives and consolidating agents. Commonly used preservatives include toluene, benzoic acid, chloroform, and thymol. Commonly used consolidating agents for proteins and enzymes include ammonium sulfate paste, sucrose, glycerin, etc., such as enzymes, substrates and coenzymes can also be added to improve their consolidation. In addition, calcium, zinc, boric acid and other solutions also have certain protective effects on certain enzymes. Nucleic acid macromolecules are usually retained in standard buffers of sodium chloride or sodium citrate.

3. The storage temperature is low, most of them are kept in the refrigerator at about 0 degrees, and some are lower, depending on different materials.

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