CN111471084A - Device for protein precipitation and method for separating and purifying target protein from protein mixed solution - Google Patents
Device for protein precipitation and method for separating and purifying target protein from protein mixed solution Download PDFInfo
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Abstract
A device for protein precipitation and a method for separating and purifying target protein from protein mixed liquor belong to the technical field of protein purification. The method aims to solve the following problems caused in the prior process of purifying protein by precipitated salt: 1) the problem of denaturation of protein molecules caused by too high local salt concentration; 2) the salting-out phenomenon occurs because the precipitated salt and the protein mixture are not uniformly mixed. The invention provides a device for fully and uniformly mixing a precipitated salt solution and a protein mixed solution, which is used for optimizing the process of purifying target protein by using the precipitated salt solution, filtering and removing the protein which is not precipitated, and simultaneously trapping the precipitated target protein in a filter membrane, thereby effectively simplifying the purification process and simultaneously being used for purifying large-volume protein. The target protein precipitated by the device is purified by an ion exchange chromatographic column in one step, so that the impurity protein can be completely removed, the target protein with the purity of more than 95 percent is obtained, and the method has extremely high economic benefit and can be used for separating and purifying the protein.
Description
Technical Field
The invention belongs to the technical field of protein purification, and particularly relates to a device for protein precipitation and a method for separating and purifying target protein from a protein mixed solution.
Background
In the process of protein purification of the protein mixed solution, when precipitated salt solids such as ammonium sulfate solids and sodium citrate solids are added into the protein mixed solution, the instantaneous local salt concentration is too high, so that the protein structure is changed and loses activity; the existing method of slowly adding the precipitated salt or grinding the precipitated salt into powder in advance can reduce the probability of protein denaturation but increase the time for purifying the protein.
Disclosure of Invention
The method aims to solve the following problems caused in the prior process of purifying protein by precipitated salt: 1) the problem of denaturation of protein molecules caused by too high local salt concentration; 2) because the salting-out phenomenon occurs when the precipitated salt and the protein mixed solution are not uniformly mixed, the invention provides a device for protein precipitation, which comprises a solvent bottle A, a solvent bottle B, a solvent bottle C, a solvent bottle D, a speed controller 1, a premixing container 2, a peristaltic device 3, a pumping pump E4, a pumping pump F6 and a filtering device 5; wherein: the solvent bottle A is used for containing protein mixed liquid; the solvent bottle B is used for containing a precipitated salt solution; the solvent bottle C is used for containing a dissolving buffer solution; the solvent bottle D is used for collecting the protein dissolving liquid obtained by filtering through the filtering device 5; the speed controller 1 is used for controlling the flow rate of liquid in the conduit; the premixing container 2 is of a three-way structure and comprises 2 feed inlets and 1 discharge outlet, wherein the 2 feed inlets are respectively connected with the discharge end of the speed controller 1, the feed end of the speed controller 1 is respectively inserted below the liquid level of the solvent bottle A and the solvent bottle B, the discharge outlet of the premixing container 2 is connected with the feed end of the peristaltic device 3, and the discharge end of the peristaltic device 3 is connected with the feed inlet of the filtering device 5 through a pumping pump E4; the feed inlet of the pump F6 is inserted below the liquid level of the solvent bottle C, and the discharge outlet of the pump F6 is connected with the feed inlet of the filter device 5; the discharge port of the filter device 5 is inserted into the solvent bottle D.
Further, the peristaltic device 3 is a peristaltic tube with a wire mesh or strip structure, wherein the wire mesh or strip structure is made of iron wires or plastics.
Further, a filter screen with the aperture size of 0.22 μm is arranged on the filter device 5.
The invention also provides a method for separating and purifying target protein from protein mixed liquor by using the device, which comprises the following steps:
1) protein precipitation: filling the protein mixed solution into a solvent bottle A, filling the precipitated salt solution into a solvent bottle B, turning on a pumping pump E4, controlling the flow rate of the liquid in the catheter by using a speed controller 1, mixing the liquid in the solvent bottle A and the liquid in the solvent bottle B in a premixing container 2, and uniformly mixing the mixed solution by using a peristaltic device 3; then the mixed solution is filtered by a filtering device 5, and the target protein which is precipitated is obtained on the filtering device 5; the volume ratio of the protein mixed solution to the precipitated salt solution is (1: 1) - (1: 4); the precipitation salt solution is selected from one of ammonium sulfate salt solution, sodium citrate salt solution, potassium citrate salt solution and potassium phosphate salt solution, and the concentration of the precipitation salt solution is 2-4M;
2) dissolving protein: opening a pump F6, pumping the dissolving buffer solution from the solvent bottle C, dissolving the target protein precipitated on the filtering device 5, and collecting the protein dissolving solution into a solvent bottle D;
3) then purifying to obtain the target protein.
Further limiting, the volume ratio of the protein mixed liquor and the precipitated salt solution in the step 1) is 1: and 3, the precipitated salt solution is an ammonium sulfate salt solution or a sodium citrate salt solution, and the concentration is 3M.
Further defined, the dissolving buffer in the step 2) is selected from one of phosphate buffer, MES buffer and HEPES buffer, and the ratio of the using volume of the dissolving buffer to the volume of the protein mixed liquor is 2: 1.
further defined, the purification in step 3) is performed by using an ion exchange chromatography column.
Further defined, the ion exchange chromatography column is a cation exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a weak anion exchange chromatography column, or a composite ion exchange chromatography column.
The composite ion exchange column has one of high salt resistance, size exclusion or hydrophobic effect and the like and has an ion exchange function.
Further limited, the ion exchange chromatographic column is a composite type ion exchange chromatographic column Capto MMC or Captoadhere.
Further limiting, if the pH value of the buffer solution is larger than the isoelectric point pI of the target protein, and the target protein is negatively charged, selecting an anion exchange chromatographic column; if the pH value of the buffer solution is less than the isoelectric point pI of the target protein, the target protein is positively charged, and a cation exchange chromatographic column is selected.
Advantageous effects
1. In the existing method for purifying target protein by using precipitated salt, the precipitated salt solid is usually slowly added into a protein mixed solution, and after the precipitated salt is completely dissolved, the solution is kept stand for 2 hours and is centrifuged at high speed to obtain the target protein with precipitate. The method can easily change the structure of the target protein and is time-consuming. The invention provides a device for fully mixing a precipitated salt solution and a protein mixed solution and a method for purifying target protein from the protein mixed solution.
2. When the protein is precipitated, the invention firstly prepares a precipitation salt solution with a certain concentration, mixes the protein mixed solution and the precipitation salt solution according to a certain proportion and fully mixes the solution uniformly, so that salt ions and protein molecules compete for water molecules, thereby destroying hydration membranes on the surface of the protein, reducing the solubility of the protein and fully precipitating the protein from the solution. Can avoid protein denaturation. And purifying the precipitated protein by a chromatographic column to obtain the protein with higher purity.
3. When a protein is purified by using a chromatographic column, purification by means of a tag, such as adding a His tag to both ends of the target protein, is usually performed by using a nickel column. But the latter would involve an enzymatic process to remove the tag, adding virtually no purification burden. The protein purification method adopted by the invention does not need subsequent label cutting, can effectively reduce the cost and simplify the purification process.
In conclusion, the purification method is based on the precipitation of the mixture of the one-step full-automatic precipitated salt solution and the protein mixed solution, namely the precipitated salt purification and the one-step ion exchange chromatographic column purification, and the process of purifying the target protein by using the precipitated salt solution is optimized by using an automatic device, so that the protein which is not precipitated can be filtered and removed, and meanwhile, the precipitated target protein is trapped in a filter membrane, and the purity of the target protein is improved; the protein on the filter membrane is dissolved by using the dissolving buffer solution, so that the function of cyclic and efficient utilization can be achieved. In addition, the dissolved protein is purified by one-step column again, so that a target protein solution with the purity of 99.1 percent can be obtained, the purification process is effectively simplified, and the method can be used for purifying large-volume protein and has extremely high economic benefit.
Drawings
FIG. 1 is a schematic diagram of an apparatus for protein precipitation, wherein 1 is a speed controller, 2 is a pre-mixing vessel, 3 is a peristaltic device, 4 is a draw pump E4 for controlling the flow rate of a tube connected to the peristaltic device, 5 is a filtration device, 6 is a draw pump F6 for controlling the flow rate of a solution tube in a bottle C containing a solvent.
FIG. 2 shows SDS-PAGE shows the optimal volume ratio of ammonium sulfate precipitated proteins, where M represents protein marker (KDa) L S represents protein mixture, and 1/1, 1/2, 1/3, and 1/4 represent different volume ratios of protein mixture to ammonium sulfate salt solution, respectively.
FIG. 3 shows SDS-PAGE shows the optimal volume ratio of the addition of the sodium citrate precipitated proteins, wherein M represents protein marker (KDa), L S represents the protein mixture, and 1/1, 1/2, 1/3 and 1/4 represent different volume ratios of the protein mixture to the sodium citrate solution, respectively.
Fig. 4 shows a comparison of the effect of ammonium sulfate precipitation and sodium citrate precipitation on protein purification by SEC-HP L C, where the abscissa is elution time (min), and the ordinate is UV 280nm (mau), UV 260nm (mau), L S represents the result of liquid phase analysis of the protein mixture without precipitation treatment, L S > ammonium sulfate precipitation represents the result of liquid phase analysis after the protein mixture is treated with ammonium sulfate salt solution, and L S > sodium citrate precipitation represents the result of liquid phase analysis after the protein mixture is treated with sodium citrate solution.
FIG. 5 shows the results of separation and purification of a sample after ammonium sulfate precipitation treatment by a Capto MMC chromatography column, with the abscissa representing the elution volume (ml), the left ordinate representing UV 280nm (mAU), and the right ordinate representing the Conductivity (mS/cm).
FIG. 6 shows SDS-PAGE analysis of peak fractions collected after Capto MMC purification, where M represents protein marker (KDa) and L S is protein mixture.
FIG. 7 analysis of SEC-HP L C after purification by Capto MMC, analysis of the peak components collected on the abscissa is elution time (min) and on the ordinate is UV 280nm (mAU) and UV 260nm (mAU).
Detailed Description
The present invention relates to the nouns or terms:
a pump is pumped: the device comprises two pumping pumps which are named as a pumping pump E and a pumping pump F respectively because the controlled pipelines are different.
Protein dissolving solution: refers to a solution obtained by dissolving a protein with a buffer.
Dissolving buffer solution: refers to the buffer used to solubilize proteins.
A speed controller: a device for controlling the flow rate of a liquid, such as a liquid flow meter or liquid flow controller, acts to control the flow rate of liquid flowing through a conduit.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention. The experimental procedures in the examples, unless otherwise specified, were carried out using techniques conventional in the art. If no special indication is made, all chemical reagents are commercial conventional analytical pure reagents, and if no special indication is made, the related detection instruments are conventional instruments or equipment in the field.
The target protein of the present invention may be an antibody, such as a nanobody, and the following examples illustrate the purification of a nanobody VHH.
Example 1. apparatus for protein purification.
The invention provides a device for protein precipitation, which comprises a solvent bottle A, a solvent bottle B, a solvent bottle C, a solvent bottle D, a speed controller 1, a premixing container 2, a peristaltic device 3, a pumping pump E4, a pumping pump F6 and a filtering device 5, wherein the solvent bottle A, the solvent bottle B, the solvent bottle C and the solvent bottle D are arranged in sequence; wherein: the solvent bottle A is used for containing protein mixed liquid; the solvent bottle B is used for containing a precipitated salt solution; the solvent bottle C is used for containing a dissolving buffer solution; the solvent bottle D is used for collecting the protein dissolving liquid obtained by filtering through the filtering device 5; the speed controller 1 is used for controlling the flow rate of liquid in the conduit; the premixing container 2 is of a three-way structure and comprises 2 feed inlets and 1 discharge outlet, wherein the 2 feed inlets are respectively connected with the discharge end of the speed controller 1, the feed end of the speed controller 1 is respectively inserted below the liquid level of the solvent bottle A and the solvent bottle B, the discharge outlet of the premixing container 2 is connected with the feed end of the peristaltic device 3, and the discharge end of the peristaltic device 3 is connected with the feed inlet of the filtering device 5 through a pumping pump E4; the feed inlet of the pump F6 is inserted below the liquid level of the solvent bottle C, and the discharge outlet of the pump F6 is connected with the feed inlet of the filter device 5; the discharge port of the filter device 5 is inserted into the solvent bottle D.
The creeping device 3 can be a creeping pipe internally provided with a wire mesh or a strip-shaped structure made of iron wires or plastics and used for slowing down the flowing speed of liquid so as to fully and uniformly mix the mixed liquid flowing through the pipe, and the filtering device 5 is provided with a filtering net with the aperture size of 0.22 mu m.
When the device works, the pumping pump E4 is started, the protein mixed solution in the solvent bottle A and the precipitated salt solution in the solvent bottle B are mixed according to a certain proportion and are pumped into the premixing container 2 through the speed controller 1 for premixing, the premixed solution can be fully and uniformly mixed in the process of passing through the peristaltic device 3, the uniformly mixed solution is pumped onto the filtering device 5 with a filtering net of 0.22 mu m through a conduit for filtering, the protein with the precipitation is intercepted on the filtering device, the filtrate is discarded, the pumping pump F6 is started, the dissolving buffer solution in the solvent bottle C is pumped onto the filtering device 5, the target protein on the filtering net is washed, the precipitation containing the target protein is dissolved, the filtrate (namely, the protein dissolved solution) is collected into the solvent bottle D, and the target protein is obtained after purification.
Example 2 determination of optimal parameters for protein purification using the apparatus described in example 1. The method comprises the following specific steps:
1) the method comprises the steps of preparing a 3M ammonium sulfate solution, placing the solution in a solvent bottle B, placing a phosphate buffer solution (20mM PB, pH7.0) in a solvent bottle C, placing a protein mixed solution in the solvent bottle A, setting the flow rate of a conduit communicated with the solvent bottle A to be 1M L/min through a pipeline speed controller 1, setting the flow rate of a conduit communicated with the solvent bottle B to be 1M L/min through the pipeline speed controller 1, mixing the protein mixed solution and the ammonium sulfate solution in a volume ratio of 1: 1, turning on a drawing pump E4, setting a total flow rate to be 2M L/min, drawing the liquid in the solvent bottle A and the liquid in the solvent bottle B into a premixing container 2 for premixing, fully mixing the liquid in a peristaltic tube 3, then passing through a filtering device 5 with the pore size of 0.22 mu M, retaining the precipitated protein on the filtering device 5, filtering supernatant into a solvent bottle D, stopping mixing the protein mixed solution with the 3M ammonium sulfate solution after the protein in the solvent bottle A is drawn out, turning off the drawing pump E4, replacing the supernatant into a solvent bottle D, and weighing the total volume of the solvent bottle A.
2) And (3) dissolving the protein, namely opening a pump F6 to enable the phosphate buffer solution to wash the precipitated protein on the filter membrane, and filtering the protein into a solvent bottle D to obtain a precipitation dissolving solution in which a protein mixed solution and an ammonium sulfate solution are mixed according to a volume ratio of 1: 1, wherein the volume of the phosphate buffer solution used in the process is a m L.
Turning on a pump E4, setting a total flow rate of 1.5m L/min, setting flow rates of pipelines for communicating the solvent bottle A and the solvent bottle B to be 1m L/min and 0.5m L/min respectively, turning off the pump E4 until all liquid in the solvent bottle A is pumped out, wherein the mixing ratio of the protein mixed solution and the ammonium sulfate salt solution is 1: 2 (volume ratio), the solvent bottle D is supernatant with the mixing ratio of 1: 2 (volume ratio), and liquid in the solvent bottle D is poured into the solvent bottle A.
Then, the pump F6 is turned on, and the phosphate buffer solution pumped a m L dissolves the precipitated protein on the filter membrane to obtain a precipitate dissolved solution in which the protein mixed solution and the ammonium sulfate salt solution are mixed in a ratio of 1: 2 (volume ratio).
Turning on a pump E4, setting a total flow rate of 1.0m L/min, setting flow rates of pipelines for communicating the solvent bottle A and the solvent bottle B to be 0.75m L/min and 0.25m L/min respectively, turning off the pump E4 until all liquid in the solvent bottle A is pumped out, wherein the mixing ratio of the protein mixed solution and the ammonium sulfate salt solution is 1: 3 (volume ratio), and meanwhile, supernatant with the mixing ratio of 1: 3 is obtained in the solvent bottle D, and pouring the liquid in the solvent bottle D into the solvent bottle A.
Then, the pump F6 is turned on, and the phosphate buffer solution pumped a m L dissolves the precipitated protein on the filter membrane to obtain a precipitate dissolved solution in which the protein mixed solution and the ammonium sulfate salt solution are mixed in a ratio of 1: 3 (volume ratio).
Opening a pump E4, setting a total flow rate of 1.25m L/min, setting flow rates of pipelines for communicating a solvent bottle A and a solvent bottle B to be 1m L/min and 0.25m L/min respectively, closing the pump E4 until all liquid in the solvent bottle A is pumped, wherein the mixing ratio of the protein mixed liquid and the ammonium sulfate salt solution is 1: 4 (volume ratio), the supernatant in the solvent bottle D is 1: 4 (volume ratio), pouring the liquid in the solvent bottle D into the solvent bottle A, then opening a pump F6, and pumping a m L phosphate buffer solution to dissolve precipitated protein on a filter membrane to obtain a precipitation solution in which the protein mixed liquid and the ammonium sulfate salt solution are mixed according to the ratio of 1: 4 (volume ratio).
And (3) respectively mixing the protein mixed solution and the ammonium sulfate salt solution according to the proportion of 1: 1. 1: 2. 1: 3. 1: 4 (volume ratio) the supernatant and the precipitate obtained after filtration were subjected to SDS-PAGE (FIG. 2). It is seen from the figure that the mixing ratio between the protein mixture and the ammonium sulfate salt solution is in the range of 1: 1 (volume ratio), the target protein is mainly in the supernatant; mixing the components in a mixing ratio of 1: 2 (volume ratio) with the target protein in both the supernatant and the precipitate; mixing the components in a mixing ratio of 1: 3 (volume ratio) is mainly in the precipitate, and the supernatant is almost free; mixing the components in a mixing ratio of 1: no target protein was observed in both supernatant and pellet at 4 (vol/vol). To sum up, in the protein mixture and ammonium sulfate salt solution 1: 1 (volume ratio), removing the precipitate, leaving supernatant, and adding ammonium sulfate solution into the supernatant again until the mixing ratio is 1: 3, at this point the supernatant was discarded and the pellet was left.
3) The obtained precipitation solution can be purified to obtain high-purity target protein.
Example 3. method for precipitating nanobody VHH using sodium citrate solution, according to the following steps:
1) preparing 3M sodium citrate salt solution, placing phosphate buffer solution (20mM PB, pH7.0) in a solvent bottle B, placing a protein mixed solution in the solvent bottle A, setting the flow rate of a pipeline communicated with the solvent bottle A to be 1M L/min through a pipeline speed controller 1, setting the flow rate of a pipeline communicated with the solvent bottle B to be 1M L/min through the pipeline speed controller 1, mixing the protein mixed solution and the sodium citrate salt solution in a volume ratio of 1: 1, opening a pumping pump E4, setting the total flow rate to be 2M L/min, pumping the liquid in the solvent bottle A and the liquid in the solvent bottle B into a premixing container 2 for premixing, fully mixing the liquid in a peristaltic tube 3, then passing through a filtering device 5 with the pore size of 0.22 mu M, retaining the protein precipitated on the filtering device 5, filtering supernatant into a solvent bottle D, stopping mixing the protein solution with the 3M sodium citrate salt solution after the protein in the solvent bottle A is pumped, closing the pumping pump E4, replacing the supernatant in the solvent bottle A, and weighing the total volume of a M L of the solvent mixed solution.
2) And (3) dissolving the protein, namely opening a pump F6 to enable the phosphate buffer solution to wash the precipitated protein on the filter membrane, and filtering the protein into a solvent bottle D to obtain a precipitation dissolving solution in which a protein mixed solution and a citric acid sodium salt solution are mixed according to a volume ratio of 1: 1, wherein the volume of the phosphate buffer solution used in the process is a m L.
Turning on a pump E4, setting a total flow rate of 1.5m L/min, setting flow rates of pipelines for communicating a solvent bottle A and a solvent bottle B to be 1m L/min and 0.5m L/min respectively, turning off the pump E4 until all liquid in the solvent bottle A is pumped, turning on the pump F6 when the mixing ratio of the protein mixed solution and the citric acid sodium salt solution is 1: 2 (volume ratio), turning on a pump D to dissolve precipitated protein on a filter membrane by phosphoric acid buffer solution of pumping a m L, and obtaining a precipitated solution of the protein mixed solution and the citric acid sodium salt solution according to the ratio of 1: 2 (volume ratio).
Opening a pumping pump E4, setting a total flow rate of 1m L/min, setting flow rates of pipelines for communicating a solvent bottle A and a solvent bottle B to be 0.75m L/min and 0.25m L/min respectively, and closing the pumping pump E4 until all liquid in the solvent bottle A is pumped, wherein the mixing ratio of the protein mixed solution and the sodium citrate solution is 1: 3 (volume ratio), and meanwhile, supernatant with the mixing ratio of 1: 3 is in the solvent bottle D, pouring the liquid in the solvent bottle D into the solvent bottle A, then opening a pumping pump F6, and pumping a m L phosphate buffer solution to dissolve precipitated protein on the filter membrane to obtain a precipitated dissolved solution in which the protein mixed solution and the sodium citrate solution are mixed according to the ratio of 1: 3 (volume ratio).
Turning on a pump E4, setting a total flow rate of 1.25m L/min, setting flow rates of pipelines for communicating a solvent bottle A and a solvent bottle B to be 1m L/min and 0.25m L/min respectively, turning off the pump E4 until all liquid in the solvent bottle A is pumped, turning on the pump F6, and turning on a phosphate buffer solution for pumping a m L to dissolve precipitated protein on a filter membrane to obtain a precipitated solution of the protein mixed solution and the sodium citrate solution in a ratio of 1: 4 (volume ratio).
And (3) respectively mixing the protein mixed solution and the citric acid sodium salt solution according to the ratio of 1: 1. 1: 2. 1: 3. 1: 4 (volume ratio) the supernatant and the precipitate obtained by filtration after mixing were subjected to SDS-PAGE (FIG. 3), and it was seen that the ratio of the mixture was 1: 1 (volume ratio) to 1: 2 (volume ratio), the protein of interest was present in both the supernatant and the pellet, in a mixing ratio of 1: 3 (volume ratio) is mainly in the precipitate, and the supernatant is almost free; mixing the components in a mixing ratio of 1: no band of the target protein was observed in both the supernatant and the precipitate at 4 (volume ratio), indicating that the target protein was almost absent. The optimal mixing ratio of the protein mixed solution to the sodium citrate is 1: and 3, discarding the supernatant, and reserving a precipitate dissolving solution.
SEC-HP L C (size exclusion high performance liquid chromatography) is used for analyzing components of samples treated by the ammonium sulfate precipitated salt solution and the sodium citrate precipitated salt solution (figure 4), the purity of the samples treated by the sodium citrate precipitated salt solution reaches 61.9 percent, the recovery rate reaches 61.4 percent, the host protein residual quantity is 39427ppm, and the DNA residual quantity is 12602ppb, while the purity of the samples treated by the ammonium sulfate precipitated salt solution reaches 69.1 percent, the recovery rate is 96.7 percent, the host protein content is 38504ppm, and the DNA residual quantity is 35253 ppb.
3) The obtained precipitation solution can be purified to obtain high-purity target protein.
Example 4. method for separating and purifying a protein of interest from a protein mixture using the apparatus described in example 1.
The preferred method of the invention is that the volume ratio of the protein mixed solution to the 3M ammonium sulfate salt solution is 1: 1 hour, discarding the precipitate, leaving the supernatant, and adding ammonium sulfate solution into the supernatant until the volume ratio reaches 1: at time 3, the supernatant was discarded, leaving a precipitate. Mixing under the action of a sucking pump, fully contacting and uniformly mixing in a subsequent creeping pipe, and then passing through a filtering device with a filtering function, so that the precipitated protein is intercepted in the filtering device. The precipitate on the filter unit was then dissolved using a phosphate buffer at pH7.0 and the dissolved precipitate was filtered out again. And then purifying the sample dissolved with the precipitated protein by a composite ion exchange chromatographic column Capto MMC to finally obtain the high-purity target protein.
The following method for purifying the nano antibody VHH by combining ammonium sulfate precipitation and a composite ion chromatographic column Capto MMC is carried out according to the following steps:
1) depositing protein, namely placing 200M L of protein mixed liquor containing a nano antibody VHH into a solvent bottle A, placing 3M ammonium sulfate solution into a solvent bottle B, placing 20mM phosphate buffer solution into a solvent bottle C, setting the flow rate of a pipeline in the solvent bottle A through the pipeline speed controller 1 to be 1M L/min, setting the flow rate of a pipeline in the solvent bottle B through the pipeline speed controller 1 to be 1M L/min, mixing the protein mixed liquor and the ammonium sulfate solution in a volume ratio of 1: 1, starting a pumping pump E4, setting the total flow rate to be 2M L/min, pumping the liquid in the solvent bottle A and the liquid in the solvent bottle B into a pre-mixing container 2, fully mixing the liquid in a peristaltic pipe 3, then passing through a filtering device 5 with the pore size of 0.22 mu M, stopping the protein precipitated on the filtering device 5, filtering supernatant into a solvent bottle D, pumping the protein mixed liquor in the solvent bottle A, closing the pumping pump E4, and immediately pouring supernatant into the solvent bottle D.
2) Protein solubilization the pump F6 was turned on to allow the phosphate buffer to solubilize the precipitated proteins on the filter and filtered into the solvent bottle D, in which 400m L of phosphate buffer was consumed while discarding the resulting protein solubilization solution.
Opening a pump E4, regulating the flow rate to 2M L/min, setting the flow rate of a pipeline communicated with a solvent bottle A to be 1M L/min through the pipeline speed controller 1, setting the flow rate of a pipeline communicated with a solvent bottle B to be 1M L/min through the pipeline speed controller 1, closing the pump E4 after all supernatant in the solvent bottle A is pumped out, opening a pump F6, setting the flow rate to be 1M L/min, pumping 400M L phosphate buffer solution to dissolve precipitate on a filtering device 5, thus obtaining the precipitate dissolving solution of which the ratio of the protein mixed solution to the 3M ammonium sulfate solution is 1: 3 (volume ratio), and filtering the precipitate dissolving solution to be purified by using a filter membrane of 0.22 mu M.
3) Then purifying to obtain the target protein.
The target protein with high purity is difficult to obtain by only one-step precipitation method, and the target protein with high purity is obtained by one-step column purification on the basis of salt precipitation. The column purification at this step often selects an ion exchange chromatography column, which is a cation exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a weak anion exchange chromatography column or a complex ion exchange chromatography column. The cation chromatographic column is negatively charged, can adsorb protein with positive charge in the filler, and then elutes the protein with positive charge from the filler under the action of increasing the concentration of salt ions. The anion chromatographic column is positive charge and can adsorb protein with negative charge, and the protein adsorbed on the filler can be eluted by increasing the concentration of salt ions. The specific selection of the cation chromatographic column or the anion chromatographic column can be determined according to the isoelectric point of the target protein and the pH value of a buffer solution in which the target protein is positioned, if the pH value of the buffer solution is greater than the isoelectric point pI of the target protein, the target protein is negatively charged, and the anion exchange chromatographic column is selected; if the pH value of the buffer solution is less than the isoelectric point pI of the target protein, the target protein is positively charged, and a cation exchange chromatographic column is selected.
In this example, a composite ion exchange chromatography column Capto MMC was used for purification.
The method comprises the following steps: and (4) further purifying the precipitate dissolving solution obtained in the previous step by utilizing a Capto MMC chromatographic column.
Firstly, starting an NGC Chromatography System, adjusting the pump to a 'Manual L oad L oop' Mode, putting a A, B pump sample inlet pipe into deionized water, setting the Mode to be Isoratic, setting the% B to be 50, switching to a flow rate setting interface, adjusting the flow rate to be 2m L/min, clicking 'Start', and flushing for 5 min.
Then, the flow rate setting interface was opened, the flow rate was set to 1m L/min, the upper pressure limit was set to 40psi, and "Apply" was clicked, a Capto MMC was mounted on the NGC Chromatography System, and 10 column volumes (10m L) were equilibrated with deionized water.
Third, open the flow rate setting interface, click "Stop", put A, B pump into the 20mM PB, 2M NaCl (pH 7.0) solution, flow rate still set at 1M L/min, Mode set at Isocaltic,% B set at 50, click "Start", activate the column with 20mM PB-2M NaCl (pH 7.0) solution using 20 column volumes (20M L).
Fourth, open the flow rate setting interface, click "Stop", put A, B pump into 20mM PB, 1M NaCl (pH 7.0) solution, flow rate still set at 1M L/min, Mode set at Isocaltic,% B set at 50, click "Start", equilibrate the column with 20mM PB, 1M NaCl (pH 7.0) solution using 20 column volumes (20M L).
Fifth, open the flow rate setting interface, click "Stop", place the A pump into 20mM PB (pH 7.0) solution, still set the flow rate at 1m L/min, Mode set at Isocratic,% B set at 0, click "Start", equilibrate the column with 20mM PB solution using 50 column volumes (50m L).
And sixthly, opening a flow rate setting interface, clicking 'Stop', putting the sample tube A into the precipitation dissolving solution to be purified, setting the flow rate to be 1m L/min, setting the Mode to be Isoratic, setting the% B to be 0, and clicking 'Start'.
Seventh, when all of the purified pellet lysate was loaded onto the column, the flow rate setting interface was opened, and "Stop" was clicked, the A-pump was placed into a 20mM PB (pH 7.0) solution, with the flow rate still set at 1m L/min, Mode set at Isoratic,% B set at 0, and "Start" was clicked, and the column was equilibrated with 20mM PB solution using 50 column volumes (50m L).
Eighth, open the flow rate setting interface, click "Stop", place the A pump into a 20mM PB (pH 7.0) solution, still set at 1m L/min, Mode set for Gradient,% B set for Start 0End 90, and Duration60min click "Start".
Ninth, open the flow setting interface, still set at 1M L/min, Mode set at Isocratic,% B set at 100, click "Start" and wash the column 10 column volumes (10M L) with 20mM PB, 1M NaCl (pH 7.0).
Tenth, open flow interface, click "Stop", place B into 1M NaOH solution, Mode set to Isocratic,% B set to 100, "Start", wash column 10 column volumes with 1M NaOH (10M L).
The tenth step, open flow interface, click "Stop", place B loading tube in 20% ethanol solution, Mode set to Isocratic,% B set to 100, click "Start", store column with 20% ethanol, rinse 20 column volumes (20m L), unload and store Capto MMC, open flow interface, click "Stop", close NGC chromatography system purification results see fig. 5 and 6.
After the protein mixed solution is subjected to ammonium sulfate salt solution precipitation and Capto MMC composite chromatographic column method purification, a single target protein band (shown as a peak 2 in figure 5 and figure 6) can be seen, SEC-HP L C analysis is carried out on a sample of which the peak is eluted at the same time, the result is shown as a peak 2 in figure 7, the purification effect is good, relatively pure target protein can be separated, the residual amount of host DNA is reduced from 12602ppb to 10154ppb, and the residual amount of host protein is reduced from 38427ppm to 373 ppm.
The device for protein precipitation is also suitable for precipitation treatment of other proteins; meanwhile, the method for separating and purifying the target protein from the protein mixed solution is also suitable for separating and purifying other proteins.
Claims (10)
1. A device for protein precipitation is characterized by comprising a solvent bottle A, a solvent bottle B, a solvent bottle C, a solvent bottle D, a speed controller (1), a premixing container (2), a peristaltic device (3), a suction pump E (4), a suction pump F (6) and a filtering device (5); wherein: the solvent bottle A is used for containing protein mixed liquid; the solvent bottle B is used for containing a precipitated salt solution; the solvent bottle C is used for containing a dissolving buffer solution; the solvent bottle D is used for collecting the protein dissolving liquid obtained by filtering through the filtering device (5); the speed controller (1) is used for controlling the flow rate of liquid in the conduit; the premixing container (2) is of a three-way structure and comprises 2 feed inlets and 1 discharge outlet, wherein the 2 feed inlets are respectively connected with the discharge end of the speed controller (1), the feed end of the speed controller (1) is respectively inserted below the liquid level of the solvent bottle A and the solvent bottle B, the discharge outlet of the premixing container (2) is connected with the feed end of the peristaltic device (3), and the discharge end of the peristaltic device (3) is connected with the feed inlet of the filtering device (5) through a pumping pump E (4); a feed inlet of the pump F (6) is inserted below the liquid level of the solvent bottle C, and a discharge outlet of the pump F (6) is connected with a feed inlet of the filtering device (5); the discharge port of the filtering device (5) is inserted into the solvent bottle D.
2. Device according to claim 1, characterized in that the peristaltic means (3) are peristaltic tubes of wire mesh or strip-like structure with wire or plastic material inside.
3. The device according to claim 1, characterized in that the filter means (5) is provided with a filter mesh having a pore size of 0.22 μm.
4. A method for separating and purifying a target protein from a protein mixture by using the device of any one of claims 1 to 3, comprising the steps of:
1) protein precipitation: putting the protein mixed solution into a solvent bottle A, putting the precipitated salt solution into a solvent bottle B, turning on a pumping pump E (4), controlling the flow rate of the liquid in a conduit by using a speed controller (1), mixing the liquid in the solvent bottle A and the liquid in the solvent bottle B in a premixing container (2), and uniformly mixing the mixed solution by using a peristaltic device (3); then the uniformly mixed solution is filtered by a filtering device (5), and target protein which is precipitated is obtained on the filtering device (5); the volume ratio of the protein mixed solution to the precipitated salt solution is (1: 1) - (1: 4); the precipitation salt solution is selected from one of ammonium sulfate salt solution, sodium citrate salt solution, potassium citrate salt solution and potassium phosphate salt solution, and the concentration of the precipitation salt solution is 2-4M;
2) dissolving protein: opening a pump F (6), pumping the dissolving buffer solution from the solvent bottle C, dissolving the target protein precipitated on the filtering device (5), and collecting the protein dissolving solution into a solvent bottle D;
3) then purifying to obtain the target protein.
5. The method according to claim 4, wherein the volume ratio of the protein mixture solution and the precipitated salt solution in the step 1) is 1: and 3, the precipitated salt solution is an ammonium sulfate salt solution or a sodium citrate salt solution, and the concentration is 3M.
6. The method according to claim 4, wherein the lysis buffer in step 2) is selected from one of phosphate buffer, MES buffer and HEPES buffer, and the ratio of the volume of the lysis buffer to the volume of the protein mixture is 2: 1.
7. the method according to claim 4, wherein the purification in step 3) is performed by using an ion exchange chromatography column.
8. The method of claim 7, wherein the ion exchange chromatography column is a cation exchange chromatography column, an anion exchange chromatography column, a weak cation exchange chromatography column, a weak anion exchange chromatography column, or a composite ion exchange chromatography column.
9. The method of claim 7, wherein the ion exchange chromatography column is a composite ion exchange chromatography column, Capto MMC or Capto adhere.
10. The method of claim 8, wherein if the pH of the buffer solution is greater than the isoelectric point pI of the target protein, and the target protein is negatively charged, selecting an anion exchange chromatography column; if the pH value of the buffer solution is less than the isoelectric point pI of the target protein, the target protein is positively charged, and a cation exchange chromatographic column is selected.
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