To isolate and purify proteins, the target protein is firstly extracted from the raw material, then a large number of impurities are removed by the method of coarse separation, and finally fine separation is carried out to obtain the target protein. In this experiment, a novel gene recombinant human TNF is used as an example to elucidate the extraction and preliminary isolation of recombinant protein TNF.
Operation method
Ammonium sulfate salting out
Principle
To isolate and purify proteins, the target protein is firstly extracted from the raw material, then a large number of impurities are removed by the method of coarse separation, and finally fine separation is carried out to obtain the target protein. This experiment elucidates the extraction and preliminary isolation of recombinant protein TNF by taking novel gene recombinant human TNF as an example.The extraction of TNF is the process of lysing the fermented bacterium, in certain conditions and solutions, so that the extracted TNF is fully released, and the extracted TNF components in the mixture are collected by centrifugation. The extracted solution containing TNF contains a large number of heterogeneous proteins, as the solubility of proteins in aqueous solution mainly depends on the number of water molecules on the surface of the protein molecule, i.e., the degree of hydrophilic groups on the surface of proteins and water molecules to form a hydration film and the charge, when neutral salts such as ammonium sulfate are added to the protein solution, due to the affinity of neutral salts for water molecules is greater than that of proteins, so that the protein molecules around the The hydration membrane is weakened or disappears, and the protein solubility is reduced. At the same time, due to the addition of neutral salts, the ionic strength of the protein solution is changed, and the protein surface charge is neutralized in large quantities, which leads to the reduction of the protein solubility even more, so that the protein molecules are aggregated with each other and precipitation occurs. Different proteins will form precipitates in different concentrations of salt due to the differences in their electrically charged and hydrophilic properties. Based on this principle, a mixture of proteins can be coarsely separated. In this experiment, most of the heterogeneous proteins were removed by ammonium sulfate hydrolysis, which led to the initial purification of TNF.
Materials and Instruments
Novel Recombinant Human TNF Move I. Reagent Preparation For more product details, please visit Aladdin Scientific website.
Sucrose TRIS Hydrochloric acid Disodium ethylenediaminetetraacetic acid Sodium hydroxide Sodium hydroxide STE solution Dnase I Sodium deoxycholate solution
Dialysis bags Stirring rods Sterilization pots Electronic balance Beaker Weighing paper Medicine spoon Measuring cylinder Beaker Ice maker
1. STE (25% sucrose 10 mmol/L Tris-HCl pH 8.5 1 mmol/L EDTA)Sucrose 250 g1 mol/L Tris.HCL 10 ml0.5 mol/L EDTA 2 ml, add water to 1 000 ml115℃, 20 min autoclave sterilization
2. 1 mol/L MgCl2 Take MgCl2 203.30 g and add water to 1 000 ml
3. Tris-HCL pH 8.5Take Tris 121.14 gAdjust pH to 8.5 with HCL and add water to 1,000 ml(12 mol/L HCL about 30 ml)
4. 0.5 mol/L EDTA pH 8.5Disodium EDTA 186.12 gNaOH 20 g, add water to 1,000 ml121℃, 20 min autoclave sterilization
II. Operational steps
1. Weigh the bacteria, add STE solution at 5~10 ml/g of bacteria and stir until the bacteria are completely suspended.
2. Add 0.5~1 mg/g of bacteria to the freshly prepared lysozyme solution with STE solution, stir well with a glass rod and leave it at 4℃ for 20 min. at this time, the bacterial solution is viscous.
3. Add sodium deoxycholate (DOC) solution freshly prepared from STE solution at 10 mg/g of bacteria and stir vigorously.
4. Add 5~10 ml of 1 mol/L MgCl2 solution, stir well and then add about 40 μl of Dnase I (25 mg/ml) and stir well until the bacterial solution becomes thin.
5. 15 000 rpm, centrifugation at 4°C for 30 min.
6. Take the supernatant from the centrifuge, measure its volume accurately, determine the protein concentration, pour it into a beaker placed in an ice bath, and slowly add solid ammonium sulphate while stirring to reach 50% saturation. After the solid ammonium sulfate is safely dissolved, leave it at 0℃ for 30 min.
7. Centrifuge at 15,000 rpm for 30 min at 4°C. The supernatant is centrifuged, the volume is measured and the protein concentration is determined.
8. The centrifuged supernatant was loaded into a dialysis bag and dialyzed at 4°C with agitation in pH 8.5 20 mmol/L Tris-Hcl, 1 mmol/L EDTA solution.
Add ammonium sulfate whether it is solid ammonium sulfate, or add saturated ammonium sulfate solution, adding should be added while stirring, pay special attention to ① add ammonium sulfate slowly, because too fast will cause the protein coprecipitation, add solid ammonium sulfate should be powdered ammonium sulfate beforehand, add saturated ammonium sulfate solution should be added drop by drop; ② stirring should be slow, stirring vigorously, protein solution is prone to foam, due to the surface tension effect will cause protein denaturation. Surface tension effect will cause protein denaturation.