Protein Purification Methods and Comparison of Advantages and Disadvantages

The separation and purification of proteins is more complicated. Proteins extracted from cells or proteins obtained from solutions containing proteins through precipitation, gradient centrifugation, salting out and other methods often contain impurities. These impurities must be removed while maintaining the biological characteristics of the protein. For the chemical activity, such as the catalytic activity of an enzyme, 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, which treats proteins gently, and can prepare large amounts of purified proteins with biological activity, so they are currently the most widely used technical methods.

Protein precipitation

The protein can be dissolved in water because its surface has hydrophilic amino acids. 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 buffers, and cold buffers help maintain the activity of the target protein. Ammonium sulfate fractionation is often used as the first step of protein purification in the laboratory. It can initially crudely extract proteins and remove non-protein components. The protein is relatively stable in ammonium sulfate precipitation, and the intermediate product can be stored in this state for a short period of time. At present, protein purification mostly uses this method for crude separation and translation. There are still some problems with the ammonium sulfate precipitation method in large-scale production. Ammonium sulfate is very corrosive to stainless steel appliances. Other salts such as sodium sulfate do not have this problem, but their purification effect is not as good as ammonium sulfate. In addition to salting out, proteins can also use polymers such as PEG? PEG is an inert substance that precipitates out of antifreeze. Like ammonium sulfate, it has a stabilizing effect on protein. Gradually increase the PEG concentration in the cold protein solution under slow stirring. The protein precipitation can be obtained by centrifugation or filtration. In this state, it can be stored for a long time without damage. Protein precipitation is not a good method for protein purification, because it can only achieve several times the purification effect, and we need thousands of times of purification before we can achieve our goal. The advantage is that it can free the protein from the culture medium and cell lysate mixed with proteases and other harmful impurities.

Replacement of buffer

Although changing the buffer does not improve the purity of the protein, it plays an extremely important role in the protein purification scheme. Different protein purification methods require buffers of different pH and different ionic strength. 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 are dialysis with semipermeable membranes and frequent changes of dialysis fluid to remove salt. 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. Add buffer to one side of the plate, and the protein solution to be desalted on the other side. The protein solution is pressurized by a pump on the side of the plate, so that the solution on both sides can be kept for several hours. The internal balance is achieved. 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 desalination columns on sale. The filler in the column is particles with small pores, and protein molecules cannot enter the pores. The high concentration of salt ions flows out of the column before the two are separated. Each step of protein purification will cause the loss of the target protein, especially the step of buffer balance. The protein will bind to any surface it can touch, and rapid changes in shear, foaming, and ionic strength can easily inactivate the protein.

Ion exchange chromatography

This is the most effective method among all protein purification and concentration methods. Based on the interaction between the protein and the ion exchange resin, by choosing different buffers, the same protein can be combined with the anion exchange resin (which can bind negatively charged molecules) or the cation exchange resin. There are four charged groups used in the resin: diethylaminoethyl is used for weak anion exchange resins; carboxymethyl is used for weak cation exchange resins; quaternary ammonium is used for strong anion exchange resins; It is a strong cation exchange resin. Protein is composed of amino acids, and amino acids have different total charges in different pH environments. Most proteins are negatively charged at physiological pH (pH 6-8) and need to be purified by anion exchange column. The protein will be denatured and inactivated at extreme pH. Avoid it as much as possible. Because different proteins have different charges at a certain pH, their binding power to resin is also different. As the salt concentration in the buffer increases or the pH changes, the proteins are eluted sequentially according to the strength of the binding power. 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 salt concentration gradient is almost always used to elute the ion exchange column. With the aid of the pump, the salt concentration in the buffer flowing into the column can be increased steadily. When the ionic strength can neutralize the charge of the protein, the protein will be Elute from the column. However, it is difficult to precisely control the salt concentration in industrial production, so step-by-step elution is often used instead of continuously rising salt gradients. Compared with size exclusion chromatography, ion exchange has better specificity, more parameters can be adjusted to obtain the best purification effect, and the resin is also cheaper. It is worth mentioning that even with the most precisely controlled conditions, pure protein cannot be obtained using a single method of ion exchange, and other purification steps are required.

Affinity chromatography

Affinity chromatography is based on the specific binding of the target protein to the immobilized ligand and it is retained, and other contaminant 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 monoclonal antibody binds to the target protein too strongly. It must be eluted under harsh conditions, which will inactivate the target protein and destroy the monoclonal antibody; Other proteins, such as proteases, may also destroy antibodies or bind to them non-specifically; some monoclonal antibodies will also dissociate from the resin during the purification process and be mixed into the product, and need to be removed from the final product. The affinity column is usually used in the later stage of the purification process, when the sample volume has been reduced 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 with 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 in order to be purified by this method; secondly, the GST tag has as many as 220 amino acids, and such a large tag may be Affect the solubility of the expressed protein and form inclusion bodies, which will destroy the natural structure of the protein, making it difficult to carry out structural analysis. Sometimes, even after purification, enzyme digestion to remove the GST tag may not solve the problem. Another applicable affinity purification tag is the 6-histidine tag. The imidazole side chain of histidine can bind to metal ions such as nickel, zinc and cobalt, and carries histidine under neutral and weak alkaline conditions. The target protein of the tag is bound to the nickel column and eluted with imidazole at low pH. Compared with GST, the histidine tag has many advantages. Firstly, because it has only 6 amino acids and its molecular weight is very small, it generally needs to be removed by enzyme digestion. Secondly, the protein can be purified under denaturing conditions, and it can still be used in high concentrations of urea and guanidine. Maintain binding power; in addition, the 6-histidine tag has no immunogenicity, and the recombinant protein can be directly used to inject animals without affecting immunological analysis. Although there are so many advantages, this label still has disadvantages, such as the target protein is easy to form inclusion bodies, difficult to dissolve, poor stability, and misfolding. When the nickel column is purified, the metal nickel ions are easy to 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 non-specifically adsorb the protein, which affects the purification effect. If the target protein can specifically bind to a certain carbohydrate, or a special cofactor is required, the carbohydrate or cofactor can be solid-phased into an affinity column, and the target protein can be combined with a high concentration of carbohydrate or auxiliary Factor elution.

Hydrophobic effect

Chromatography proteins are composed of hydrophobic and hydrophilic amino acids. Hydrophobic amino acids are located in the center of the protein spatial structure, away from the 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, usually the entire protein molecule is surrounded by water molecules, and hydrophobic amino acids will not be exposed. In a high salt concentration environment, the hydrophobic region of the protein will be exposed and bind to the hydrophobic ligand on the surface of the hydrophobic medium. The hydrophobicity of different proteins is different, and the magnitude of the hydrophobic force is also 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 hydrophobic force is weakened and eluted.
The selectivity of hydrophobic resins is determined by the structure of hydrophobic ligands. Commonly used linear ligands are alkyl ligands and arylligands. The longer the chain, the greater the ability to bind proteins. powerful. The choice of the ideal resin type should be based on the chemical properties of the target protein. It is not possible to choose a resin with too strong binding force. Resins with too strong binding force will be difficult to elute. Therefore, at the beginning, choose a phenyl resin with medium binding force 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 of ion exchange purification, because hydrophobic interaction chromatography loads samples at high salt concentrations, and the products obtained from ion exchange can be used without changing the buffer. The protein is eluted in the low-salt buffer, and the step of changing the buffer before the next purification is omitted, which not only saves time, but also reduces the loss of protein.

Exclusion chromatography

Also called gel filtration or molecular sieve. The packing particles of the exclusion chromatography column are porous media, and the amount of liquid that can be contained in the column surrounding the particles is called the mobile phase, also called the ineffective volume. Proteins that are too large cannot enter the pores of the particles, and can only exist in an invalid volume of solution, and will be eluted 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, their ability to diffuse into particles of a specific size and pore size is also different. Large protein molecules will be eluted first, and the smaller the molecules, the later they will be eluted. In order to obtain the best purification effect, the pore size should be selected so that the target protein can be eluted near the midpoint of the void volume and the total column volume. Size exclusion chromatography has advantages that other methods do not have. First, the protein that can be purified has a wide molecular weight range. Tosoh Biosep’s polymer resin has an exclusion limit of up to 200,000 kD. Second, the shape of the resin micropores is suitable for separating spherical proteins. , The purification process does not require organic solvents that can cause protein denaturation. 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 them. Size exclusion chromatography is never used in the early stages of the purification process, because this method requires a high degree of concentration of the sample, and the sample load can only be between 1% to 4% of the column volume. The column must be thin and long to get a good separation effect. The resin itself is also relatively expensive, so it is not suitable for large-scale industrial production.

Electrophoresis

Acrylamide gel electrophoresis is usually used to check the complexity of protein mixture samples and monitor the purification effect. The separation effect of this method is excellent, but unfortunately it is difficult to scale up to the preparation scale without loss of precision, because as the thickness of the gel increases, the thermal effect during electrophoresis will seriously interfere with the migration of the protein. In basic research, sometimes only a small amount of pure protein is needed 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 process of protein purification. It can detect which gradient of ion exchange column salt the target protein is in; it can be used to determine the rapid development of various disciplines in recent years. The demand for purification continues to grow, the existing purification methods have been improved day by day, and new purification methods have emerged one after another. Hydroxyapatite is a crystal of calcium phosphate. Due to its insufficient physical and chemical properties and poor binding ability, it is difficult to use in chromatography. Recently, Bio-Rad has improved it by increasing the ratio of calcium and phosphorus, so that spherical, porous, and stable ceramic hydroxyapatite particles are formed, with positively charged calcium ions and negatively charged phosphate ions. Can be combined with the carboxyl and amino groups of the protein respectively. By adjusting the pH value of the buffer, acidic and basic amino acids can be selectively combined with the resin, and the salt concentration of the buffer can be changed to separate the protein. Data show that using this method can separate two proteins with the same isoelectric point, molecular weight, and hydrophobicity.

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