物质分离与纯化蛋白质提取（Substance isolation and purification, protein extraction, ）

1、物质分离与纯化蛋白质提取2（Substance isolation and purification, protein extraction, 2）(1) aqueous solution extractionDilute aqueous salt and buffer system on protein stability, solubility, solvent extraction is the most commonly used protein, usually the amount is 1-5 times the volume of raw materials, extracti

2、on should be evenly stirred, in order to facilitate the dissolution of protein. The temperature of the extraction depends on the nature of the active ingredient. On the one hand, the solubility of most proteins increases with the increase of temperature; therefore, high temperature is beneficial to

3、dissolution and shorten extraction time. On the other hand, temperature increases protein denaturation and inactivation. Therefore, based on this point, proteins and enzymes are usually used under low temperature (5 degrees Celsius). In order to avoid protein degradation in the process, we can add p

4、roteolytic inhibitors (such as two isopropyl phosphoric acid, iodine acetic acid, etc.).The choice of pH and salt concentration of the extract is discussed below.1 and pH valuesProteins are enzymes with isoelectric points, and the pH value of the extract should be chosen at the pH on the sides of th

5、e isoelectric pointWithin limits. With dilute acid or alkali extraction, should prevent acid or alkali caused by protein ionizable residues changed, which leads to irreversible protein conformational changes, in general, the basic protein by liquid extraction and acidic extraction, acidic protein wi

6、th alkaline extract.2, salt concentrationDilute concentration can promote protein dissolution, called salt dissolution. At the same time, because of the combination of salt ions and proteins, the dilute salt solution has the advantage of protecting protein from denaturation. Therefore, a small amoun

7、t of neutral salt such as NaCl is added to the extraction solution, which is usually 0.15 mol. Elevated concentration is appropriate. Buffer solution often uses 0.02-0.05M phosphate and carbonate isotonic salt solution.(two) organic solvent extractionAnd some of the lipid binding nonpolar side chain

8、s of proteins and enzymes in solid or molecule, insoluble in water, dilute salt solution, dilute acid or alkali, ethanol, acetone and butanol by organic solvents, they have certain hydrophilicity, and strong lipophilicity, extract lipid extraction the ideal protein. But it must be operated at low te

9、mperatures. Butanol extraction method for extracting lipid and closely combined with the proteins and enzymes are excellent, because alcohol especially strong lipophilicity, strong ability to dissolve lecithin; two is butanol both hydrophilic and solubility in the range (10% degrees, 40 degrees for

10、6.6%) will not cause the change of enzyme inactivation. In addition, butanol has a wide range of pH and temperature, and is suitable for animal, plant and microbiological materials.Two. Separation and purification of proteinsThere are many methods for isolation and purification of proteins:(a) separ

11、ation methods based on protein solubility1. Protein salting outHave a significant effect on protein solubility in neutral salt, generally at low salt concentration with salt concentration increased, the increase of protein solubility, the salt soluble; when the salt concentration continues to rise,

12、the protein solubility decreased in different degrees and has the phenomenon that the precipitation, salting out, large quantities of salt to the protein solution, high salt ion the concentration (such as ammonium sulfate, SO4 and NH4) with hydration force is very strong, can capture the protein mol

13、ecules in the hydration layer, so that the loss, so the protein colloidal precipitation and condensation. When salting out, if the solution pH is at the isoelectric point of the protein, the effect is better. Because the size of various protein molecules and the degree of hydrophilic are different,

14、the salt concentration required for salting out is different. Therefore, the concentration of neutral salt in the mixed protein solution can be adjusted so that the proteins can be precipitated separately.The factors affecting salting out are: (1) temperature: in addition to temperature sensitive pr

15、oteins operating at low temperature (4 degrees), they can be carried out at room temperature. Generally, the temperature is low and the solubility of proteins decreases. But some proteins (such as hemoglobin, myoglobin, albumin) at higher temperature (25 degrees) than 0 degrees of solubility is low,

16、 more easily salting out. (2) pH value: most proteins in isoelectric point have the lowest solubility in concentrated salt solution. (3) protein concentration: when the protein concentration is high, the proteins that are separated are often precipitated together with other proteins (coprecipitation

17、).Therefore, before salting out, the serum should be diluted with the same amount of physiological saline, so that the protein content is 2.5-3.0%.The neutral salts usually used in protein salting include ammonium sulfate, Magnesium Sulfate, sodium sulfate, sodium chloride, sodium phosphate and so o

18、n.One of the most widely used ammonium sulfate, the utility model has the advantages of low temperature coefficient and high solubility (25 degrees when the saturated solution is 4.1M, which is 767 g / L; 0 degree of saturation solubility is 3.9M, which is 676 g / L), in which the solubility range o

19、f many proteins and enzymes can be salted out; in addition of ammonium sulfate fractionation the effect is good than other salt, is not easy to cause the protein denaturation. The pH of ammonium sulfate solution is often between 4.5-5.5, and it needs to be adjusted by sulfuric acid or ammonia when s

20、alting out with other pH values.Protein in salting out precipitation separation, protein salt needs to be removed, the common way is that the protein solution into the dialysis bag on the show (common glass paper), dialysis buffer, and continue to replace the buffer by dialysis for a longer period o

21、f time, so the best in the low temperature of. In addition, glucose gel G-25 or G-50 column method can be used to remove salt, and the time is short.2. Isoelectric precipitationThe electrostatic repulsion between the static state of protein particles and minimum solubility is minimum, all the isoele

22、ctric point of the protein is different, by regulating the pH of the solution to precipitate a protein isoelectric point to make, but it is rarely used alone, can be combined with salting out method.3, low temperature organic solvent precipitation methodWith water miscible organic solvent, methanol,

23、 ethanol or acetone, the solubility of most proteins can be reduced and precipitated. The resolution of this method is higher than that of salting out, but the protein is easy to change and should be carried out at low temperature.(two) separation method according to the difference of protein molecu

24、lar size1 、 dialysis and ultrafiltrationDialysis is separated using semipermeable membranes with different molecular sizes of protein.Ultrafiltration is the use of high pressure or force of centrifugal force, water and other small solutes through the membrane and the protein in the membrane, can cho

25、ose Lu membrane with different pore sizes by different molecular weight protein.2, gel filtration methodMolecular exclusion chromatography or molecular sieve chromatography, which is one of the most effective methods for separating protein mixtures according to their molecular size. The most commonl

26、y used filling material in the column is glucose gel (Sephadex)GED) and agarose gel (agarose, gel).(three) separation based on the charged nature of proteinsProteins in different pH environments can be separated by the charged nature and the number of charges.1 ElectrophoresisVarious proteins are se

27、parated in the same pH condition because of their different mobility in the electric field due to their different molecular weights and charges. Worthy of attention is the isoelectric focusing electrophoresis, this is the use of an amphoteric electrolyte as carrier, electrophoresis amphoteric electr

28、olyte formed by a cathode to anode of pH increased gradually with a certain gradient, when the charge of the protein in swimming, arrive at their isoelectric point pH position on the end, the method can be used for analysis and the preparation of a variety of proteins.Ion exchange chromatography, 2I

29、on exchangers include cationic exchangers (e.g. carboxymethyl cellulose; CM- cellulose) and anion exchangers (two ethyl cellulose); DEAE; FONTFACE= Song typefaceLANG= ZH-CN ), cellulose when the protein solution is separated through ion exchange chromatography, ion exchange agent with opposite charg

30、e and the protein is adsorbed on the ion exchange agent, followed by changes in pH or ionic strength to protein adsorption elution. (see chromatography for details.)(four) affinity chromatography based on ligand specificity separation methodAffinity chromatography (aflinity)Chromatography) is a very

31、 effective method for the separation of proteins, it is often separated by only one step can make a protein to be purified from the protein mixture is very complex, and high purity. This method is based on the specific, rather than covalent, combination of certain proteins and molecules called ligan

32、ds (Ligand).The basic principle is: protein existed in the form of complex mixtures in tissues or cells, each cell type contains thousands of different proteins, the protein separation and purification, (Separation) (Purification)And identification (Characterization) is an important part in biochemi

33、stry, alone or in a ready-made method is still not to move any kind of protein extracted from the mixed protein complex, therefore often take several methods together.Cell disruption1, high-speed organization: the material with mashed into a paste liquid, placed in the cylinder volume of about 1/3,

34、covering the cylinder cover, the governor dial to the slow start, after the switch to the required speed gradually accelerated. This method is suitable for animal internal organs, plants, meat, seeds and so on.2, the glass homogenate machine: first cut the tissue on the tube, and then set into the r

35、esearch lever back and forth grinding, move up and down, can be the cell crush, cell fragmentation degree is higher than the Waring Blender, suitable for less and animal tissues.3, the ultrasonic treatment method: using certain power ultrasonic treatment of cell suspension, the cell sharp shock rupt

36、ure, this method is suitable for microbial material, preparing various kinds of enzyme in Escherichia coli, used 50-100 mg / mL concentration in the cell, 1KG to 10KG frequency 10-15 minutes, the disadvantage is in the processing to produce a lot of heat, we should take corresponding measures to cool down. Sensitive