Protein precipitation technology
Protein precipitation technology
Although many different precipitation methods have been used over the past 100 years, AS precipitation has been the most widely used, especially for acidic proteins. In addition, the use of PEI precipitation is becoming increasingly popular. These two methods will be discussed in detail, followed by a brief overview of several other precipitation methods and general recommendations for handling the precipitate during the precipitation process and maximizing purification efficiency.
Operation method
Protein precipitation technology Move I. AS precipitation 1. Principle Although some other salts can also be used as precipitants, AS is the most valuable because of its outstanding characteristics. AS stabilizes the structure of proteins very well, has excellent solubility, is relatively inexpensive, is readily available in pure form, and the density of its saturated solution at 25 ℃ (4.1InoVU1O=I.235 g/cm3) is lower than that of another salting agent, dipotassium hydrogenphosphate (3 mol/L, (0=1.33 g/cm3)). A typical protein solubility curve is shown in Figure 20.1, plotted as a function of the logarithm of protein solubility corresponding to AS concentration. The curve is characterized by the fact that protein solubility increases with increasing salt concentration in regions of lower salt concentration (known as 'saltolysis') and decreases with increasing salt concentration in regions of higher salt concentration (known as 'salting out'). The second half of this curve can be described by the equation l ○ g1DS=|3-Ks(r/2). where S is the solubility of the protein in solution expressed in mg/mL; plant/2 is the ionic strength; j3 and K3 are the characteristic constants of the protein in question. ks is the slope of the salting-out curve, and 卩 is the logarithmic value of the solubility of the protein when the salting-out curve extends to an ionic strength of 〇 . In general, most proteins have similar min values, whereas p values vary considerably. Assume that the curve shown in Figure 20.1 applies to the target protein and that its concentration in the cell extract is Img/mL. The top horizontal dashed line intersects the solubilization curve at the point where the percent saturation of AS at logS=0 (s=lmg/mL) is 26%. This means that if the AS is increased to 26% saturation, all of the target proteins used will be solubilized. If the AS is increased to 32% saturation (horizontal dashed line in the middle) with l ○ gS=_1 (S=0.1 mg/mL), 90% of the target proteins will become insoluble and precipitate. A better strategy for this extraction method would be to achieve an AS of 26%? A better strategy for this extraction method is to saturate the AS to 26%? 32% percent saturation: first, saturate the AS to 26% percent saturation to selectively isolate the insoluble material, then increase the AS saturation to 32% and collect the precipitate, which will contain 90% of the target protein. Impurity proteins and cellular fractions that precipitate at 26% saturation and those that do not precipitate at 32% saturation can be removed by precipitation. It is recommended that a 10-fold dilution of the extract with buffer be considered. At this point, the initial concentration of the target protein in the extract is 0.Img/mL or logS=-l. Increasing the saturation of the AS to 32% does not result in precipitation of the target protein. In order to obtain 90% precipitation of the target protein, the saturation of the AS should be increased to 38% (horizontal dashed line below) or a 32%?38% AS saturation interval should be taken. Eventually the extract may have to be diluted and more than 10 times the previous amount of AS used to obtain the target protein. This suggests that it is important to limit the concentration of the extract. The target protein may not necessarily have a solubility profile as shown in Figure 20.1, so the appropriate AS concentration must first be determined by performing the experiments described below. There are a number of different methods for the application of AS, the most common being the addition of solid AS to the protein extract to achieve the desired percent saturation. Referring to Table 20.1, the addition of solid AS is convenient, reproducible and practical. (1) Generally, at lower percent saturation, no precipitation of the target protein occurs, whereas at higher percent saturation, more than 90% of the protein is precipitated. (2) Add solid AS to achieve lower percent saturation. Add AS carefully and slowly while stirring at high speed to avoid localized concentration exceeding the target value. Some people use a mortar and pestle to carefully grind the solid AS into a fine powder so that it can dissolve quickly. Once the AS is fully dissolved, the precipitation can be sustained for about 30 min. This is a compromise between waiting a few hours for the precipitation to slowly equilibrate, and allowing the precipitation to proceed in parallel with the purification process without delaying the process. All operations should normally be carried out in a small ice bucket or cold room. (3) Centrifuge for 10 min at 10,000 centers in a pre-cooled rotator to remove insoluble material. (4) Carefully decant the supernatant and measure its volume. Determine the mass of AS required by adding AS in descending order of percent saturation as shown in Table 20.1. Continue with rapid stirring and add the AS slowly to avoid localized high salt concentrations, then allow to settle for 30 min. (5) Centrifuge as shown in step (3). Remove as much of the supernatant as possible, which will contain 90% or more of the target protein if the precipitation has been carefully pre-tested. The protein can be dissolved in a suitable buffer, dialyzed, desalted and diluted, and then used in the next purification step. Generally, a 10% increase in AS saturation can precipitate 90% of the target protein, so we should limit the range of the "AS interval" to no more than 10% (e.g., if the protein is just solubilized at 30% saturation, but precipitates at 40% saturation, this is referred to as the 30%?40% AS interval). The optimal AS precipitation conditions can be determined using only a two-step centrifugation procedure, as shown in Figure 20.2. Basically, the cell extract is first placed in a container, e.g., 10 mL of sample is added to each of five centrifuge tubes. Solid AS is added to the tubes according to Table 20.1 and saturated to 20%, 30%, 40%, 50%, and 60%, respectively, and left for 30 min to precipitate the proteins, followed by centrifugation to remove insoluble material. The precipitates were 20%, 30%, 40%, 50% and 60% saturated AS precipitates, respectively. The volume of the corresponding supernatant was determined and AS was added again to increase the saturation by 10%. After further mixing, the solution was allowed to settle for 30 min and then centrifuged. The resulting five precipitates are the 20%?30%, 30%?40%, 40%?50%, 50%?60% and 60%?70% AS intervals. They were dissolved in buffer and analyzed for enzyme activity and total protein content, and SDS gel analysis was also performed if necessary. Most of the proteins that are still viable should have the majority of their viability in one of these intervals. For example, with half the viability in the 30%?40% zone and half the viability in the 40%?50% zone, the 35% to 45% zone would probably be the optimal AS concentration range. Although this experiment may seem like a tedious way to tie up the process, it is certainly a very effective way to determine the optimal conditions for enriching more proteins during the precipitation step. (1) The resulting precipitate is not firm After centrifugation, if the AS precipitate does not form firmly, it will be difficult to decant the supernatant completely. A simple solution is to extend the centrifugation time by 50%, so that the sediment at the bottom of the centrifuge tube has more time to become dense. Another reason for the formation of a loose precipitate is the presence of DNA, which increases the viscosity of the solution and thus reduces the rate of precipitate formation. If viscosity is the problem, one way to solve it is to break the cells by sonication for a longer period of time, so that the DNA is sheared into shorter fragments; another way is to use a recombinant nuclease, Benzonase (EMD/Novagen), for this purpose. (2) Formation of spherical suspensions in high concentrations of AS Because the density of high concentrations of AS is comparable to that of protein aggregates, AS precipitates may exist as suspensions after centrifugation rather than as precipitates at the bottom of the centrifuge tubes. This is most likely due to the presence of some lipids in the proteins or the presence of non-ionizing detergents that bind to the proteins and reduce their density. (3) Difficulty in following published protocols Many of the published protocols do not follow the established AS saturation (saturation at 0°C, 20°C, or 25°C) or protein concentration of the extract. It is therefore important to note that the amount of AS required for precipitation depends on the protein concentration. (4) The AS precipitation step has to be interrupted If the precipitation has to be stopped, the protein can be retained in the AS precipitation. Proteins are very stable in AS, as are suspensions or precipitates that precipitate proteins. PEI, with the trade name polyethyleneimine (polyminP), is an alkaline cationic polymer produced by BASF and used in large quantities in the textile and paper industries.PEI is an alkaline polymer obtained by polymerization of vinylimine, with the structure CH3CH2N-(-CH2CH2-NH-X-CH2CH2NH2). -The classical W-value is 700?2000, and the corresponding molecular mass of the polymer is 30OOO?90OOODa. PEI is positively charged in neutral pH solution because the pKa value of imine is 10?11. The use of ;PEI for protein separation was first reported by Boe-hringerMannheim, Zillig et al. (1970). Further examples of its use for protein purification can be found in several published reviews (Burgess, 1991; BurgessandJendrisak,1975;Jendrisak,1987;JendrisakandBurgess,1975).0 PEI can be thought of as similar to soluble DEAE cellulose. binds to negatively charged macromolecules, such as nucleic acids and acidic proteins, which in turn form a mesh of PEI, followed by rapid precipitation of the bound acidic molecules. This binding occurs stoichiometrically. Heavier precipitates can form quickly and can be collected by centrifugation at 5000r/min for 5 min. Whether or not acidic proteins bind to PEI depends on the concentration of the salt solution. At low salt concentrations (0.Imol/LNaCD), slightly acidic proteins bind to PEI and form a precipitate, but at moderate salt concentrations (0.4 mol/LNaCl), it can be eluted from the multimer and become soluble. Strongly acidic proteins bind to PEI at low salt concentrations but do not solubilize at intermediate concentrations and are eluted at high salt concentrations (○.9m ○ l/LNaCl). It should be noted that when the protein is eluted from the PEI particles, both the protein and the PEI itself become soluble. Therefore, PEI needs to be removed from the protein before returning to a low salt concentration (see sections 3.2?3.5 of this chapter). There are 3 different strategies for using PEI precipitation as follows. Strategy A: PEI precipitation is used at high salt concentrations (Imol/LNaCl). This method precipitates nucleic acids, while almost all of the protein remains in the supernatant. Strategy B (for neutral or basic proteins): PEI precipitation at 0.Imol/LNaCl removes nucleic acids and acidic proteins. The target protein is retained in the supernatant. Strategy C (for acidic proteins such as RNA polymerase): This scheme, which will be discussed in more detail below, is based on the method of Burgess and Jendrisak (1975) and improved by Burgess and Knuth (1996). (1) Prepare a 10% (V/V) [5% (m/V)] PEI stock solution. PEI(WV) is often present as a 50% (m/V) viscous liquid (PEI from MPBiochemicals, with a relative molecular mass of Mw=50000? 100000, other sources such as Sigma and Aldrich with Mw=750000 can also be used). Take 10 mL of PEI, dilute to 70 mL with double-distilled water H2O, and adjust the pH with concentrated hydrochloric acid (3.8?4.0 mL) to 7.9. Finally, dilute to a final volume of 100 niL with double-distilled water H2O. The storage solution is stable in a cold room or at room temperature. It should be noted that some companies offer PEI solutions that have been diluted I:1 with distilled water H2O to reduce the viscosity of the solution and to facilitate its preparation. The concentration is only 25% (m/\0. (2) The cells were crushed by sonication in 30 mL of buffer containing 50 mmol/LTris-Ha (pH 7.9), 5% glycerol, 0.1 mmol/LEDTA, 0.Immol/LDTT, and 0.15 mol/LNaCl? .cW cells (approximately 3 g of cell sediment). Cell debris was removed by centrifugation at 15000r/min for 15 min. All operations were performed in an ice bath. (3) The PEI precipitation assay was applied to specific systems (described below). For example, add pH 7.910% (V/V) PEI to a final concentration of 0?3% (V/V) and mix for 5 min until a thick white precipitate forms. (4) Centrifuge at 5000r/min for 5 min. Note: Do not centrifuge too hard or the precipitate will not be resuspended easily. Discard the 0.3% PEI supernatant and save for further analysis. Dry the precipitate for 1?2 min to remove as much of the supernatant as possible. (5) Sufficiently resuspend the 0.3% PEI precipitate with 30 mL of the above buffer containing 0.4 mol/L NaCl. If conditions permit, a TissueTearor homogenizer (BioSpecProducts, Inc. Cat#985370-07) can be used, which can resuspend the precipitate well, effectively physically washing out proteins trapped within the precipitate and eluting slightly acidic proteins that are weakly bound to the precipitate PEI. Leave for 5 min, followed by centrifugation at 5000r/min for 5 min and decantation of 0.4mol/LNaCl buffer. (6) Resuspend the 0.4 mol/L NaCl precipitate well with 30 mL of the above buffer containing 0.9 mol/L NaCl. This elution contains more acidic proteins (e.g., RNA polymerase), but at the same time leaves nucleic acids in the precipitate (nucleic acids can be washed out with I.6 md/L NaCl). Allow to stand for approximately 5 min, mix, and centrifuge at 15,000 r/min for 10 min. (7) For the 0.9 mol/L NaCl eluate, add solid AS to saturate to 60% (3.61 g per 10 mL). Continue mixing for at least 30 min to form a precipitate. Centrifuge at 15,000 r/min for 10 min and dehydrate the precipitate for 5 min. The precipitate contains the proteins precipitated by the AS and almost all of the PEI will remain in the supernatant. However, traces of PEI may be trapped in the precipitate, which usually does not interfere with the following operations. If it is necessary to remove these traces more completely, the precipitate can be resuspended in a buffer containing 60% saturated AS and centrifuged again. This procedure generally results in a 6-fold purification of RNA polymerase from other proteins, with recoveries greater than 90%, and removal of almost all of the nucleic acid in 1?2 h. The following procedure is recommended for the removal of PEI. (1) It should be emphasized that removal of PEI from the 0.9 mol/L NaCl eluate is necessary. If only dilution or dialysis is used to achieve a low salt concentration, proteins will still bind to PEI and precipitate again. (2) We found that PEI precipitation occurred even in the presence of 1% TritonX-100. (3) Unlike AS precipitation, the same amount of PEI needs to be added when the extract is diluted 10-fold with buffer (e.g., for a normal extract, 0.3% PEI precipitates the target proteins well, whereas for a 10-fold dilution of the extract, the same precipitation can be achieved by adding only 0.03% PEI, of course, in a volume 10 times that of the extract). This suggests that PEI can tightly bind the acidic component, essentially titrating it. Since we cannot predict how much PEI will be needed to precipitate a particular acidic protein, or what concentration of salt will be used to elute the protein from the PEI, it is recommended that a simple PEI precipitation and elution experiment be performed first (Burgess and Jendrisak, 1975; Burgess and Knuth, 1996). Basically, the steps of this experiment are described below. Six 200 samples were loaded into six microcentrifuge tubes and 10% (VAZ) PEI was added to achieve final concentrations of 0%, 0.l%, 0.2%, 0.3%, 0.4%, and 0?5% (V/V), respectively. The microcentrifuge tubes were mixed at high speed with Imin, and the supernatant was analyzed by enzyme activity or SDS gel electrophoresis, and, if desired, by WesternBlot to detect the minimum amount of PEI required to precipitate all target proteins, which we assumed to be 0.3%. Next, 6 microcentrifuge tubes, each containing a small amount of 0.3% PEI precipitate, were prepared as described above and 200 JL1L of buffer containing 0 mol/L, 0.2 mol/L, 0.4 mol/L, 0.6 mol/L, 0.8 mol/L, and I.0 mol/L NaCl was added. Resuspend well. Allow to stand for 15 min, centrifuge, and analyze the target proteins in the supernatant as described above. The solution with the highest salt concentration that does not elute any proteins is used as the wash solution, and the solution with the salt concentration that elutes all target proteins is used as the elution solution. 3.5 Example: Precipitation of DNA-bound basic proteins by PEI Recently, researchers (Duellman and Burgess, 2008) sought to purify a strongly basic protein expressed by coZi, Epstein-Barrvirus nuclear antigen1 (EBNA1). We performed PEI precipitation experiments at 0.1 mol/L NaCl to investigate whether nucleic acids and acidic proteins could be precipitated, and the basic EBNAl was retained in the supernatant (strategy B). Unexpectedly, EBNA1 could be precipitated when a small amount of PEI (0.15%) was added, but it could not be precipitated when more PEKO (4%) was added, suggesting that EBNAl had already been bound to the DNA, and was then precipitated together with the DNA. However, in the presence of a higher concentration of PEI, PEI preferentially binds to DNA and displaces EBNAl. We found that it is possible to precipitate EBNAl with 0.15% PEI, wash it with 0.3 mol/L NaCl, and elute it with 0_8 mol/L NaCl. We found that 0.15% PEI could be used for precipitation, 0.3 mol/L NaCl for washing, and 0_8 mol/L NaCl for elution, which could remove the nucleic acid quickly while enriching EBNAl. This chapter has already focused on AS precipitation and PEI precipitation. The following section will briefly discuss other methods used for protein precipitation, which have been described in detail in a large body of literature [see Englard and Seifter (1990); Ingham (1990); Scopes (1994)]. Precipitation with organic solvents such as ethanol and acetone has been used for more than 100 years, but is best known for the classic work of Cohen and Edsall, who used it for the isolation of human serum proteins. The precipitation method must be operated at a low temperature to avoid denaturation of the proteins. Proteins become insoluble at their isoelectric point, when they have a net charge of 〇 and the charge repulsion between protein molecules is relatively minimal, so they can easily approach each other. Proteins are not soluble at very low ionic strengths, so it is also possible to perform isoelectric point precipitation at very low salt concentrations or in the absence of salt. In this method, the cell extract is heated to a certain temperature, at which point many proteins will be denatured and precipitate, but the target proteins will remain soluble because they are more stable. This method can be used especially to purify expressed thermophilic bacterial enzymes, specifically by heating the cell extract to a high enough temperature that almost all of the ? :? 0^ proteins are denatured and precipitated, while the heat-stabilized enzyme is retained in solution. This method can be reviewed in Ingham (1990). (1) In the washing or elution stage, it is very important to resuspend the protein precipitate sufficiently. Although the precipitate appears to be very firm, a large amount of supernatant is trapped in it and adheres to the walls of the centrifuge tube. As mentioned previously, try to dry the precipitate as much as possible to remove the supernatant present. If the precipitate is larger than the supernatant, resuspension with 10 times the volume of a suitable buffer is recommended to remove the supernatant proteins trapped in the precipitate. For example, when precipitating with 40% saturated AS, the precipitate can be resuspended with 40% saturated AS and then centrifuged again. Washing the PEI precipitate is a very useful step, and a homogenizer similar to the TissueTearor is recommended to break up the precipitate and form a homogeneous suspension. If the resuspension is not adequate, then the washing step will not be effective in removing nucleic acids and the separation will be reduced accordingly. (2) Avoid foam formation during mixing. If air is mixed into the protein solution, it will promote the oxidation of the protein and cause protein denaturation at the air-water interface. For more product details, please visit Aladdin Scientific website.
