Separation and purification of proteins is more complicated. Proteins extracted from cells or proteins obtained by precipitation, gradient centrifugation, and salting out of solutions containing proteins often contain impurities. To remove these impurities, it is necessary to maintain the biological properties of the protein For scientific activities, such as the catalytic activity of enzymes, it is necessary to formulate corresponding strategies and adopt different methods according to different proteins. Electrophoresis and chromatography are more commonly used methods, especially chromatography. The protein treatment is relatively gentle, and a large number of biologically active purified proteins can be prepared, so it is currently the most widely used technical method.
Protein is soluble in water because of the hydrophilic amino acids on its surface. If the ionic strength of the solution is particularly high or low at the isoelectric point of the protein, the protein tends to precipitate out of the solution. Ammonium sulfate is the most commonly used salt for protein precipitation, because it has good solubility in cold buffer, and cold buffer is helpful to maintain the activity of the target protein. Ammonium sulfate fractionation is often used as the first step in laboratory protein purification. It can crudely extract proteins and remove non-protein components. Proteins are more stable in ammonium sulfate precipitation, and intermediate products can be preserved in this state for a short period of time. Currently, protein purification mostly uses this method for crude separation. There are still some problems in the ammonium sulfate precipitation method on large-scale production. Ammonium sulfate is very corrosive to stainless steel appliances. Other salts such as sodium sulfate do not have this problem, but its purification effect is not as good as ammonium sulfate. In addition to salting out, can proteins use polymers such as PEG? Precipitated with antifreeze, PEG is an inert substance, which has the same stabilizing effect on protein as ammonium sulfate, and gradually increases the concentration of PEG in the cold protein solution under slow stirring. Protein precipitation can be obtained by centrifugation or filtration. In this state, long-term storage without damage. Protein precipitation is not a very good method for protein purification, because it can only achieve a few times the purification effect, and we need thousands of times of purification before reaching the goal. The advantage is that the protein can be released from the culture medium and cell lysate mixed with protease and other harmful impurities.
Although changing the buffer does not improve protein purity, it plays an extremely important role in protein purification protocols. Different protein purification methods require different pH and different ionic strength buffers. If you use ammonium sulfate to precipitate the protein, there is no doubt that the protein is in a high-salt environment, and you need to find a way to desalinate. The available methods include using semi-permeable membrane dialysis and removing salt by frequently changing the dialysis fluid. This method is acceptable. But it takes several hours, usually overnight, and it is difficult to use in large-scale purification. The new type of equipment sandwiches the dialysis membrane between two plates. One side of the plate is added with buffer solution, and the other side is added with the protein solution to be desalted, and the protein solution is pressurized by a pump. The internal balance is reached. If the pressure on the protein solution is increased, more water and salt can be forced into the dialysate through the dialysis membrane to achieve the purpose of protein concentration. There are also desalting columns for sale. The packing in the column is particles with small pore size. Protein molecules cannot enter the pores, and the salt ions flow out of the column before high concentration, so that the two are separated. Each step of protein purification will cause the loss of the target protein, especially the step of buffer equilibration. The protein will bind to any surface it can contact, and shear forces, foaming, and rapid changes in ionic strength can easily inactivate the protein.
This is the most effective method among all protein purification and concentration methods. Based on the mutual charge interaction between the protein and the ion exchange resin, by choosing different buffers, the same protein can be combined with either anion exchange resin (which can bind negatively charged molecules) or cation exchange resin. There are four kinds of charged groups used in the resin: diethylaminoethyl for weak anion exchange resin; carboxymethyl for weak cation exchange resin; quaternary ammonium for strong anion exchange resin; methylsulfonate Yu strong cation exchange resin. Proteins are composed of amino acids, which have different total charges in different pH environments. Most proteins are negatively charged at physiological pH (pH 6 to 8) and need to be purified with an anion exchange column. At extreme pH, the protein will be denatured and inactivated. It should be avoided as much as possible. Due to the different charge numbers of different proteins at a specific pH, the binding force with the resin is also different. As the salt concentration in the buffer increases or the pH changes, the protein is eluted in order according to the strength of the binding force. In industrial production, it is more to change the salt concentration than to change the pH value, because the former is easier to control. In the laboratory, the ion exchange column is almost always eluted with a salt concentration gradient. With the aid of the pump, the salt concentration in the buffer flowing into the column can be steadily increased. When the ionic strength can neutralize the charge of the protein, the protein is trapped. Elute from the column. However, in industrial production, the salt concentration is difficult to control precisely, so the stepwise elution is often not sufficient for a continuously increasing salt gradient. Compared with exclusion chromatography, ion exchange has better specificity, more parameters can be adjusted to obtain the best purification effect, and the resin is cheaper. It is worth mentioning that even with the most precisely controlled conditions, pure protein cannot be obtained by a single method of ion exchange, and other purification steps are required.
Affinity chromatography is based on the specific binding of the target protein to the solid-phase ligand, and other contaminated proteins will flow through the column. The problem with this method is that the monoclonal antibody is very expensive and needs to be purified first; the binding capacity of the monoclonal antibody to the target protein is too strong. It must be eluted under harsh conditions, which will inactivate the target protein and destroy the monoclonal antibody; mixture Other proteins such as proteases may also destroy antibodies or bind them non-specifically; some monoclonal antibodies will also dissociate from the resin during the purification process and mix into the product, which also needs to be removed from the final product. Affinity columns are usually used late in the purification process, when the specimen volume has shrunk and most of the impurities have been removed. Glutathione S-transferase (Glutathione S-transferase, GST) is one of the most commonly used affinity chromatography purification tags. Recombinant proteins with this tag can be purified by cross-linked glutathione chromatography media, but This method has the following disadvantages: first, the GST on the protein must be properly folded to form a spatial structure that binds to glutathione to be purified by this method; second, the GST tag has up to 220 amino acids, such a large tag may be It affects the solubility of the expressed protein and forms inclusion bodies, which will destroy the natural structure of the protein and make it difficult to analyze the structure. Sometimes, even after purification, the GST tag may not be removed by digestion. Another applicable affinity purification tag is the 6-histidine tag. The imidazole side chain of histidine can affinity bind metal ions such as nickel, zinc, and cobalt, with histidine under neutral and weak alkaline conditions. The target protein of the tag is combined with a nickel column and is eluted with imidazole at low pH. Compared with GST, histidine tags have many advantages. First, because there are only 6 amino acids, the molecular weight is very small, and generally need to be removed by enzyme digestion. Second, the protein can be purified under denaturing conditions, and it can still be used in high concentrations of urea and guanidine. Maintain the binding force; the other 6 histidine tags are not immunogenic, and the recombinant protein can be directly used to inject animals without affecting immunological analysis. Although there are so many advantages, this tag still has shortcomings, such as the target protein is easy to form inclusion bodies, difficult to dissolve, poor stability and misfolding. During the purification of the nickel column, the metal nickel ions easily fall off and leak into the protein solution. Not only will the amino acid side chain of the target protein be destroyed by oxidation, but the column will also specifically adsorb the protein, affecting the purification effect. If the target protein can specifically bind to a certain carbohydrate, or need a special cofactor, the carbohydrate or cofactor can be solid-phased to make an affinity column. After binding, the target protein can be used with a high concentration of carbohydrate or cofactor Factor elution.
Chromatographic proteins are composed of hydrophobic and hydrophilic amino acids. Hydrophobic amino acids are located in the center of the protein spatial structure, away from water molecules on the surface. Hydrophilic amino acid residues are located on the surface of the protein. Since hydrophilic amino acids attract many water molecules, the entire protein molecule is usually surrounded by water molecules, and hydrophobic amino acids are not exposed. In the environment of high salt concentration, the hydrophobic region of the protein will be exposed and bind to the hydrophobic ligand on the surface of the hydrophobic medium. Different proteins have different hydrophobicity, and the magnitude of the hydrophobic force is different. By gradually reducing the salt concentration in the buffer to wash the column, when the salt concentration is very low, the protein returns to its natural state, and the weakened hydrophobic force is eluted.
The selectivity of the hydrophobic resin is determined by the structure of the hydrophobic ligand. Commonly used linear ligands are alkyl ligands and arylligands. The longer the chain, the more the ability to bind proteins Strong. The choice of the ideal resin type should be based on the chemical properties of the target protein. The resin with too strong binding force cannot be selected, and the resin with too strong binding force will be difficult to elute, so the phenyl resin with moderate binding force should be selected at the beginning to explore the conditions . To make it easier to choose the right medium, Amersham Biosciences has launched a hydrophobic interaction resin selection kit, which includes 5 different resins for comparison. Hydrophobic chromatography is very suitable as the next step in ion exchange purification, because hydrophobic interaction chromatography is loaded at a high salt concentration, and the product obtained from ion exchange can be used without changing the buffer. The protein is eluted in the low-salt buffer, and the step of replacing the buffer before the next purification is omitted, which not only saves time but also reduces protein loss.
Also called gel filtration or molecular sieve. The packed particles of an exclusion chromatography column are porous media. The amount of liquid that can be contained in the column surrounding the particles is called the mobile phase, also known as the invalid volume. Proteins that are too large cannot enter the pores of the particles, and can only exist in an ineffective volume of solution, which will elute from the column at the earliest and have no purification effect on this part of the protein. Due to the different molecular sizes of various proteins, the ability to diffuse into pores of a certain size is also different. Large protein molecules will be eluted first. The smaller the molecule, the later it will elute. In order to obtain the best purification effect, the pore size should be selected so that the target protein can elute near the midpoint of the void volume and the total column volume. Exclusion chromatography has advantages that other methods do not have. First, the molecular weight of the protein that can be purified is wide. Tosoh Biosep’s polymer resin has an exclusion limit of 200,000 kD. Second, the shape of the resin pores is suitable for the separation of spherical proteins. Also, no organic solvents that can cause protein denaturation are needed during the purification process. It should be noted that some proteins are not suitable for purification by gel filtration, because the resin used in this technology is slightly hydrophilic, and proteins with higher charge density are easily adsorbed on it. Exclusion chromatography is never used in the early stage of the purification process, because this method requires highly concentrated specimens, and the sample load can only be between 1% and 4% of the column volume. The column must be thin and long to obtain good separation results. The resin itself is relatively expensive, and it is not suitable for large-scale industrial production.
Gel electrophoresis is commonly used to view the complexity of protein mixture samples and monitor the effectiveness of purification. The separation effect of this method is excellent, but unfortunately it is difficult to scale up to the preparation scale without losing accuracy, because as the thickness of the gel increases, the thermal effect during electrophoresis can seriously interfere with the swimming of the protein. In basic research, sometimes only a small amount of pure protein is required for research, such as protein sequencing, etc. At this time, electrophoretic purification is a simple and fast method. Acrylamide gel electrophoresis is also an important analysis tool in the protein purification process. It can detect the gradient of the ion exchange column salt eluent of the target protein; it can be used to determine the protein purification technology with the rapid development of various disciplines in recent years. The demand for is increasing, the existing purification methods are being improved day by day, and new purification methods are also emerging one after another. Hydroxyapatite is a crystal of calcium phosphate. Due to its unstable physical and chemical properties and poor binding ability, it is difficult to use for chromatography. Recently, Bio-Rad has improved it to increase the ratio of calcium and phosphorus to form spherical, porous, and stable ceramic hydroxyapatite particles with positively charged calcium ions and negatively charged phosphate ions. Can be combined with the carboxyl and amino groups of the protein. By adjusting the pH value of the buffer, acidic and basic amino acids can be selectively combined with this resin, and the salt concentration of the buffer can be changed to elute and separate the protein. The data shows that using this method can make two proteins with the same isoelectric point, molecular weight and hydrophobicity well .
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