What are the methods of protein separation, their characteristics are what

1. According to the molecular size of different separation and purification

Protein is a large molecular material, and different protein molecular size is different, so you can use some of the simpler methods to make protein

quality and small molecules separate, and make the protein mixture is also separated. According to the different size of protein molecules to separate the main methods are dialysis, ultrafiltration, centrifugation and gel filtration. Dialysis and ultrafiltration are commonly used methods to separate proteins. In dialysis, the mixture to be separated is placed in a dialysis bag made of a semi-permeable membrane and then immersed in dialysate for separation. Ultrafiltration is a process that utilizes centrifugal force or pressure to force water and other small molecules to pass through a semi-permeable membrane, while the proteins are retained on the semi-permeable membrane. Both methods separate protein macromolecules from small molecules dominated by inorganic salts. They are often used in conjunction with salting-out and salting-out methods, which can be utilized to remove introduced inorganic salts after salting-out or salting-out. During ultrafiltration, the surface of the membrane is easily clogged by adsorbed proteins, which slows down the ultrafiltration rate and reduces the molecular weight of the intercepted substances. Therefore, when using ultrafiltration, we should choose the appropriate membrane, and we can also choose tangential flow filtration to get more ideal results

Centrifugation is also often used in conjunction with other methods of separating proteins. Centrifugation can be used to separate proteins from impurities when they have different solubilities. For example, in the experiment of extracting proteins from rice pomace, cellulase and α-amylase were added for pretreatment, and then centrifugation was used to initially separate the useful substances from the decomposed impurities [3]. Centrifugation of proteins in a medium with a density gradient is called density gradient (zone) centrifugation. The commonly used density gradients are sucrose gradient, poly sucrose gradient and density gradients of other synthetic materials. The appropriate density gradient can be selected based on the range of densities and osmotic pressures required. Density gradient centrifugation has been used to purify Bacillus thuringiensis companion spore crystal proteins, and the products obtained were of high purity but low yield. Jiang Chen et al[6] obtained high purity B. thuringiensis companion spore crystal proteins using sodium bromide density gradient by comparing the separation effect of different density gradient media. Gel filtration, also known as gel permeation chromatography, is one of the most effective methods to separate proteins according to the different sizes of protein molecules. The principle of gel filtration is that when different proteins flow through the gel chromatography column, the molecules with larger pore size than the gel beads can not enter the mesh structure inside the beads, and are blocked outside the gel beads, and with the solvent in the gap between the gel beads to move downward and the first out of the column; on the contrary, than the molecules with smaller pore size than the gel beads after the flow out of the column. At present, the commonly used gels are cross-linked dextran gel, polyacrylamide gel and agarose gel. The use of gel filtration in the purification of mannan proteins can get very good results, and the purity identification proved that the product is a single homogeneous glycoprotein with a molecular weight of about 32 kDa, a composition of polysaccharide:protein (88:12), and a polysaccharide of mannose [1]. Gel filtration has also been used in the extraction of anticoagulant proteins to remove most of the heterogeneous proteins and small molecule impurities [7].

2. Separation and purification according to different solubility

There are many external conditions affecting the solubility of proteins, such as the pH of the solution, ionic strength, dielectric constant and temperature. However, under the same conditions, different proteins have different solubility due to their different molecular structures, according to the characteristics of the protein molecular structure, appropriate changes in the external conditions, you can selectively control the solubility of a component of the protein mixture, to achieve the purpose of the separation and purification of proteins. Commonly used methods include isoelectric point precipitation and pH adjustment, salt solubilization and salting out of proteins, organic solvent method, double aqueous phase extraction, and antiglue extraction.

Isoelectric point precipitation and pH adjustment are the most commonly used methods. Each protein has its own isoelectric point, and in the isoelectric point when the solubility is the lowest

On the contrary, some proteins in a certain pH value is easy to dissolve. Therefore, proteins can be separated and purified by adjusting the pH of the solution. Wang Hongxin et al[8] studied the tea protein extraction process found that the pH value of tea protein extraction is the best, the extraction rate reached 36-8%, the initial purification rate of 91-0%. Li Dianbao [9] in the extraction of protein from sunflower defatted meal will be adjusted to the pH value of the protein solution to 3 ~ 4, so that the target protein in the isoelectric point precipitation out. The isoelectric point precipitation method was also applied to the extraction of proteins from grape seeds. Li Fengying et al [10] measured that the isoelectric point of grape seed protein is 3-8, and they used alkaline solvent method to extract grape seed protein, and got the optimal extraction process as follows: 1×10-5mol-L-1 NaOH solution, according to the material-liquid ratio of 1:5, stirring at 40 ℃ for 40 min, and the extraction rate of grape seed protein reaches 73-78%. In addition, rice protein can also be extracted by alkaline method, and its water retention, oil absorption and foaming properties are better than those of enzymatic extraction [11]. Using acid extraction of silver carp fish protein without fishy taste, white color, protein yield of up to 90% [12].

Salt solubilization and salting out of proteins is a phenomenon in which neutral salt significantly affects the solubility of globular proteins, in which the phenomenon of increasing protein solubility is called salt solubilization, and vice versa for salting out. It should be noted that the effect of the same concentration of divalent ionic neutral salts, such as MgCl2, (NH4)2SO4 on protein solubility is much larger than that of monovalent ionic neutral salts such as NaCl, NH4Cl. In the grape seed protein extraction process in addition to alkali solubilization can also be used to extract protein using salting out method, the optimal extraction process is: 10% NaCl solution, according to the material-liquid ratio of 1:25, stirring at 30 ℃ extraction for 30 min, the protein extraction rate of 57-25% [10]. Salt precipitation is a common method for extracting immunoglobulins from blood, such as sodium polyphosphate flocculation method, ammonium sulfate salting out method, of which ammonium sulfate salting out method is widely used in production. Because ammonium sulfate is acidic in water, in order to prevent its destruction of proteins, ammonia should be used to adjust the pH value to neutral. In order to prevent the phenomenon of *** precipitation between different molecules, the content of protein samples is generally controlled at 0-2% ~ 2-0%. After the purification of proteins by salting out and salting out, neutral salts are usually removed by dialysis or gel filtration [13].

The principle of organic solvent extraction is that organic solvents (e.g., methanol, ethanol) that are miscible with water can significantly reduce the solubility of some proteins in water; and the concentration of organic solvents that cause protein precipitation varies at certain temperatures, pH values, and ionic strengths, so that controlling the concentration of organic solvents can separate and purify proteins. For example, slow addition of ethanol (-25°C) to pre-cooled culture medium at 4°C under magnetic stirring in an ice bath can precipitate ice nucleated proteins and thus purify them [14]. Since at room temperature, organic solvents not only cause protein precipitation, but also accompany denaturation. Therefore, the problem of protein denaturation can be solved to a large extent by cooling the organic solvent and then adding the organic solvent under constant stirring to prevent the local concentration from being too high. For some proteins that are more firmly bound to lipids or have more polar side chains in the molecule and are insoluble in water, they can be extracted with organic solvents such as ethanol, acetone and butanol, which have a certain degree of hydrophilicity and strong lipophilicity, and are the ideal extracting solutions. Cold ethanol separation method for immunoglobulin extraction was firstly proposed by Cohn in 1949 for the preparation of gammaglobulin. Cold ethanol method is also recommended by the WHO protocol and the Chinese biologics protocol, which not only has high resolution, good purification effect, can separate multiple plasma components at the same time, but also has the effects of bacteriostasis, clearance and virus inactivation [15].

Extraction is a commonly used method for the separation and purification of organic compounds, and biphasic extraction and antigella extraction can be used to separate proteins. Aqueous two phase extraction (ATPE) refers to the formation of a hydrophilic polymer aqueous solution into a double aqueous phase under certain conditions, due to the difference in the distribution of the separated material in the two phases, the separation can be realized, and it is widely used in biochemistry, cell biology, and biochemistry and other fields of product separation and extraction. The method can be carried out at room temperature, and the polymer in the biphasic phase also improves the stability of the proteins, resulting in higher yields. For intracellular proteins, it is necessary to crush the cells effectively. The target proteins are usually distributed in the upper phase and concentrated, while solids such as cell debris are distributed in the lower phase. Concentration of target proteins using a two-phase aqueous system is affected by the molecular weight and concentration of the polymer, solution pH, ionic strength, salt type and concentration [16].

Anti-cellulite extraction is used to extract proteins by encapsulating them with anticellulite. An antigella is a nano-sized aggregate formed when surfactants

aggregate spontaneously when dissolved in a non-polar organic solvent. The advantage of this method is that the egg

white matter is protected by the anticluster during the extraction process because it is located inside the anticluster. Cheng Shixian et al [17] then extracted proteins from soybeans using antigella extraction.

3. Separation and purification according to different charges

The methods for separating proteins according to their charges, i.e., acid-base properties, are electrophoresis and ion exchange chromatography.

Under the action of external electric field, the charged particles (such as protein molecules not in the state of isoelectric point) will move toward the electrode opposite to its electrical properties, and this

kind of phenomenon is called electrophoresis. Polyacrylamide electrophoresis is a kind of zone electrophoresis using polyacrylamide as medium, which is commonly used for separating proteins. It has the advantages of simple equipment, convenient operation, and small amount of sample. Isoelectric focusing is a high-resolution protein separation technique, which can also be used for isoelectric point determination of proteins. The separation of protein mixtures by isoelectric focusing is carried out in a medium with a pH gradient. Under the action of an external electric field, various proteins will be shifted and focused to form a narrow band at a pH gradient equal to their isoelectric point. Sun Chenzhong et al [18] studied the application of polyacrylamide electrophoresis, isoelectric focusing electrophoresis and isocratic purification electrophoresis in the separation and purification of proteins. It was found that polyacrylamide electrophoresis had low band resolution and low sample volume; isoelectric focusing electrophoresis had the highest resolution and could separate subcomponents of the same protein with the smallest sample volume; isotachophoretic purification electrophoresis had high resolution of the bands, and could separate the sample into a single component with the largest sample volume.

Ion exchange chromatography (IEC) is a chromatographic method in which the ion exchange agent is used as the stationary phase, and the separation is carried out according to the difference in binding force between the component ions in the mobile phase and the equilibrium ions on the exchange agent when they are reversibly exchanged. In ion exchange chromatography, the matrix consists of a resin or cellulose with an electrical charge. Positively charged anion exchange resins and cation exchange resins. Ion exchange chromatography can also be used for the separation and purification of proteins. When proteins are under different pH conditions, their charged status is also different. The anion-exchange matrix binds negatively charged proteins, which are retained on the chromatographic column, and the proteins adsorbed on the column are eluted by increasing the salt concentration in the eluent, and the weaker bound proteins are eluted first. On the contrary, the cation exchange matrix binds positively charged proteins, and the bound proteins can be eluted by gradually increasing the salt concentration in the eluent or raising the pH of the eluent. Li Quanhong et al [19] applied ion exchange chromatography to the purification of proteins from apple juice concentrate. In addition, ion exchange chromatography was also used for the extraction of anticoagulant proteins [7].

4. Separation and purification using specific affinity for ligands

Affinity chromatography is an effective purification method established by using the unique ability of protein molecules to recognize their ligand molecules (i.e., biological affinity). It usually requires only one step to separate the target protein from a complex mixture, and the purity is quite high. The application of affinity chromatography requires knowledge of the structure and biological properties of the purified material in order to design the best separation conditions. In recent years, affinity chromatography has been widely used in the isolation and purification of target proteins, especially vaccines, especially in the isolation and purification of fusion proteins, affinity chromatography plays a pivotal role, because the fusion proteins have the ability to bind specifically [20]. Affinity chromatography is also widely used in the isolation and purification of genetically engineered subunit vaccines [21]. Fan Jiye et al [22] used chitosan affinity chromatography to extract peptidase with specific activity of 71 428 BAEE-mg-1 and purification recovery of 62-5%. The method is low-cost, inexpensive adsorbent, high mechanical strength, strong anti-pollution ability, small non-specific adsorption, repeated use, wide applicability, and stable product quality.