Extraction of tissue nucleoprotein by traditional methods

Nucleoprotein: /view/264309.htm

Glycoprotein: /view/ 184084.htm

Lipoprotein: /view/ 1333 14.htm

Similarities and differences: The chemical essence is protein, but glycoprotein has sugar side chains, which play different roles in different positions.

Protein's separation method and principle;

Separate and purifying accord to different molecular sizes.

? Protein is a macromolecular substance, and its molecular size is different from that of protein. We can make protein by some simple methods.

Mass and small molecular substances are separated, and protein mixture is also separated. According to the different molecular sizes of protein, the main separation methods are dialysis, ultrafiltration, centrifugation and gel filtration.

2. Separate and purify according to different solubility.

There are many external conditions that affect the solubility of protein, such as pH value, ionic strength, dielectric constant and temperature. However, under the same conditions, different protein have different solubility because of their different molecular structures. According to the characteristics of the molecular structure of protein, the solubility of a component in protein mixture can be selectively controlled by appropriately changing the external conditions, so as to achieve the purpose of separating and purifying protein. Commonly used methods include isoelectric point precipitation and pH adjustment, salt dissolution and salting-out of protein, organic solvent method, aqueous two-phase extraction method, reverse micelle extraction method and so on.

Isoelectric precipitation and pH adjustment are the most commonly used methods. Each protein has its own isoelectric point, where it is most soluble.

Low; On the contrary, some protein can be easily dissolved at a certain pH value. Therefore, protein can be separated and purified by adjusting the pH value of the solution. Wang Hongxin and others [8] studied the extraction technology of tea protein, and found that when the pH value was 0, the extraction effect of tea protein was the best. The initial purification yield was 965438 0.0%. When Li Dianbao [9] extracted protein from sunflower defatted meal, he adjusted the pH value of protein solution to 3~4 to precipitate the target protein at the isoelectric point. Isoelectric precipitation method is also suitable for extracting protein from grape seeds. Li et al. [10] measured the isoelectric point of grape seed protein at 3.8. The protein in grape seeds was extracted by alkali liquor, and the optimum extraction process was obtained: NaOH solution was/kloc-0 /×10-5 mol L-1,the ratio of material to liquid was 1∶5, and the extraction rate of grape seeds protein reached 73.78%. In addition, the water holding capacity, oil absorption and foaming ability of rice protein extracted by alkaline method are better than those extracted by enzymatic method [1 1]. The silver carp protein extracted by acid method has no fishy smell and white color, and the protein yield is as high as 90%[ 12].

Salt dissolution and salting-out in protein are phenomena that neutral salts significantly affect the solubility of globular proteins. Among them, the phenomenon that protein solubility increases is called salt dissolution, and vice versa. It should be pointed out that the same concentration of divalent ion neutral salts, such as MgCl2 and (NH4)2SO4, has much greater influence on the solubility of protein than monovalent ion neutral salts, such as NaCl and NH4Cl. In the process of extracting grape seed protein, protein can be extracted with salt solution or alkali solution. The optimum extraction process is: 10%NaCl solution, 1∶25 solid-liquid ratio, stirring at 30℃ for 30min, and the extraction rate of protein is 57.25% [10]. Salting out method is a common method to extract immunoglobulin from blood, such as sodium polyphosphate flocculation method and ammonium sulfate salting out method, among which ammonium sulfate salting out method is widely used in production. Because ammonium sulfate is acidic in water, in order to prevent its harm to protein, the pH value is adjusted to neutral with ammonia water. In order to prevent precipitation between different molecules, the content of protein samples is generally controlled at 0.2% ~ 2.0%. After salting-out and salting-out purification of protein, neutral salts are usually removed by dialysis or gel filtration [13].

The principle of organic solvent extraction method is that organic solvents (such as methanol and ethanol) that are miscible with water can significantly reduce the solubility of some protein in water; Moreover, under certain temperature, pH value and ionic strength, the concentration of organic solvents causing protein precipitation is different. Therefore, protein can be separated and purified by controlling the concentration of organic solvents. For example, under magnetic stirring in an ice bath, slowly adding ethanol (-25℃) to the pre-cooled culture solution at 4℃ can precipitate the ice nucleation protein, thus purifying the ice nucleation protein [14]. Because at room temperature, organic solvents will not only cause protein precipitation, but also be accompanied by denaturation. Therefore, it is usually necessary to cool the organic solvent, and then add the organic solvent under constant stirring to prevent the local concentration from being too high, so that the problem of protein denaturation can be solved to a great extent. Some protein are strongly bound to lipids or have many polar side chains in their molecules, which are insoluble in water. They can be extracted with organic solvents such as ethanol, acetone and butanol. These solvents have certain hydrophilicity and strong lipophilicity, and are ideal extraction solutions. The extraction of immunoglobulin by cold ethanol separation was first proposed by Cohn in 1949 for the preparation of gamma globulin. Cold ethanol method is also recommended by WHO and China biological products regulations at present, which not only has high resolution and good purification effect, but also can separate various plasma components at the same time, and has the functions of bacteriostasis, elimination and virus elimination [15].

Extraction is a common method to separate and purify organic compounds. Aqueous two-phase extraction and reverse micelle extraction can be used to separate protein. Aqueous two-phase extraction (ATPE) refers to the formation of aqueous two-phase by hydrophilic polymer solution under certain conditions, which can be separated due to the different distribution of separated substances in the two phases, and is widely used in the separation and extraction of products in biochemistry, cell biology and biochemical engineering. The method can be carried out at room temperature, and the polymer in aqueous two-phase can also improve the stability of protein with high yield. For intracellular proteins, it is necessary to break cells effectively first. The target protein is often distributed in the upper phase and concentrated, and solid substances such as cell fragments are distributed in the lower phase. The aqueous two-phase system was used to concentrate the target protein, which was influenced by the molecular weight and concentration of polymer, pH value of solution, ionic strength, salts and concentration [16].

The reverse micelle extraction method is to extract protein by wrapping protein with reverse micelles. Reverse micelles are used as surfactants.

Nanoscale aggregates formed by spontaneous aggregation when nonpolar organic solvents are dissolved. The advantage of this method is that in the process of extraction, eggs

White material is protected by the reverse micelle because it is located inside the reverse micelle. Cheng Shixian et al [17] used reverse micelle extraction to extract protein from soybean.

3. Separation and purification according to different charges.

According to the charge of protein, that is, the nature of acid and alkali, there are two methods to separate protein: electrophoresis and ion exchange chromatography.

Under the action of external electric field, charged particles (such as protein molecules not in isoelectric point state) will move to the opposite electrode, which

This phenomenon is called electrophoresis. Polyacrylamide electrophoresis is a kind of zonal electrophoresis with polyacrylamide as the medium, which is often used to separate protein. Its advantages are simple equipment, convenient operation and less sample consumption. Isoelectric focusing is a high-resolution protein separation technique, which can also be used to determine the isoelectric point of protein. Isoelectric focusing separation of protein mixture was carried out in a medium with pH gradient. Under the action of external electric field, all kinds of protein will move to the pH gradient equal to its isoelectric point and concentrate to form a narrow strip. Sun et al [18] studied the application of polyacrylamide electrophoresis, isoelectric focusing electrophoresis and isokinetic purification electrophoresis in the separation and purification of protein. The results showed that the resolution of polyacrylamide electrophoresis bands was low and the sample size was not high. The resolution of isoelectric focusing electrophoresis is the highest, and the subcomponents of the same protein can be separated with the least sample amount. Isokinetic purification electrophoresis zone has high resolution, and the sample can be divided into single components, with the largest sample addition.

Ion exchange chromatography (IEC) is a chromatographic method, which uses ion exchanger as stationary phase, and separates according to the difference of binding force between component ions in mobile phase and equilibrium ions on exchanger during reversible exchange. In ion exchange chromatography, the matrix consists of charged resin or cellulose. Anion exchange resin with positive charge; On the contrary, it is cation exchange resin. Ion exchange chromatography can also be used for the separation and purification of protein. When protein is at different pH values, its charged state is also different. The anion exchange matrix combined with negatively charged protein remains on the chromatographic column. By increasing the salt concentration in the eluent, protein adsorbed on the chromatographic column was eluted, and protein with weak binding was eluted first. On the contrary, the cation exchange matrix is combined with positively charged protein, and the combined protein can be eluted by gradually increasing the salt concentration in the eluent or increasing the pH value of the eluent. Li et al [19] applied ion exchange chromatography to the purification of protein in concentrated apple juice. In addition, ion exchange chromatography is also used to extract anticoagulant proteins [7].

4. Separation and purification are carried out by using the specific affinity of ligands.

Affinity chromatography is an effective purification method based on the unique recognition ability (biological affinity) of protein molecules to its ligand molecules. Usually, only one step is needed to separate the target protein from the complex mixture, and the purity is quite high. In order to design the best separation conditions, it is necessary to understand the structure and biological characteristics of the purified substances by affinity chromatography. In recent years, affinity chromatography technology has been widely used in the separation and purification of target proteins, especially vaccines, especially fusion proteins, because fusion proteins have specific binding ability [20]. Affinity chromatography is also widely used in the separation and purification of genetically engineered subunit vaccines [2 1]. The specific activity of aprotinin extracted by Fan et al. [22] chitosan affinity chromatography reached 765,438+0,428 baeemg-65,438+0, and the purification recovery rate reached 62.5%. The method has the advantages of low cost, low adsorbent price, high mechanical strength, strong anti-pollution ability, small nonspecific adsorption, reusability, wide applicability and stable product quality.