CN112830954B - Efficient protein separation and purification method and device based on uniform mixing-liquid removal process - Google Patents

Abstract

The invention relates to a method and a device for efficiently separating and purifying protein based on a uniform mixing-liquid removing process. The motor drives the inner cylinder to rotate to generate centrifugal force to load Ni ²⁺ The resin and the protein purification solution are subjected to solid-liquid separation, so that the aim of low-cost and high-efficiency preparation of the protein is fulfilled. The present invention can be applied to Ni-carrying ²⁺ The separation and purification of the specific combination of the resin and the His label protein can also be suitable for the separation and purification of charged protein and ion exchange particles through electrostatic adsorption. The cost of separating and purifying protein is far lower than that of chromatography. Can overcome the defect that the chromatography can not process the high-titer recombinant protein. The traditional chromatography method generally needs 5 to 6 hours for separating and purifying protein, and the invention can greatly shorten the separation and purification time which can be realized only by 30min. The invention relates to a device.

Description

Efficient protein separation and purification method and device based on uniform mixing-liquid removal process

Technical Field

The invention belongs to the field of protein separation and purification, and relates to a method and a device for efficiently separating and purifying protein based on a blending-liquid removal process ²⁺ The solid-liquid separation of the resin and the protein purification solution realizes the purpose of low-cost and high-efficiency preparation of the protein.

Background

The biomedical industry is currently the most promising industry in the market, and the current investment industry agrees to consider that the investment of biomedical items is very potential. Protein drugs have been developed vigorously because of their advantages of high activity, strong specificity, low toxicity, clear biological function studies, etc. Protein drugs in the pharmaceutical market include genetically engineered drugs, polypeptide drugs, monoclonal antibodies, recombinant genetically engineered antibodies, recombinant vaccines, and the like. The invention of the recombinant insulin is an important step for overcoming and treating diabetes mellitus of human beings, and the recombinant insulin is the first recombinant protein drug. In 1982, likky, USA, firstly put recombinant insulin on the market, and marked the birth of the first recombinant protein drug.

Protein separation and purification are currently a key step that restricts the development of protein drugs, and the methods for protein separation and purification are generally determined according to the physicochemical properties of proteins, such as size, shape, charge, isoelectric point, specific binding, and the like. The industrial protein preparing process includes upstream fermentation to express high titer target protein and downstream separation to purify high purity target protein. The existing protein separation and purification methods comprise chromatography, membrane separation, precipitation salt precipitation, organic solvent precipitation, crystallization, magnetic separation and the like. Chromatography is the most commonly used separation and purification process for industrial production of proteins. However, chromatography has a number of problems: 1. chromatography requires much time to bind the target protein, and therefore chromatography brings about many time cost problems. 2. The bottleneck problem faced by chromatography is that the prior art cannot handle high upstream titer protein products. 3. Since chromatography is performed by fixing a resin to a column, there are many dead spaces in the column where a protein solution cannot contact the resin, and the resin is not sufficiently used. Therefore, in order to solve the above problems, the present invention proposes and designs a novel method and apparatus for protein purification with low cost and high efficiency.

Chromatographic techniques were used for the separation of organic pigments and are therefore also referred to as chromatographic techniques, and have been developed for use in protein separation and purification. In 1975 immobilized metal ion affinity chromatography began to develop, typically transition metal ion Cu ²⁺ 、Zn ²⁺ 、Ni ²⁺ Chelating on resin, and combining the metal ions with the specific amino acid on the surface of protein through electrostatic adsorption, coordination or covalent bond. Purification of the recombinantly expressed protein is generally by immobilized metal ion affinity chromatography. As the His label is expressed in prokaryotic escherichia coliThe system has good compatibility in the transcription and translation process and the expression product has good solubility, so that the solubility of the target protein in the solution is not changed. The His label has low influence on the expressed biological function of the target protein and low immunogenicity, and the produced protein can be applied without cutting off the His label. The use of His-tag expression systems in recombinant protein isolation procedures can reduce this process of tag removal. At present, when a recombinant protein expression system is constructed, a 6 × His tag is generally added to the front end or the tail end of a target gene segment. Then IPTG induces and expresses the target protein containing His tag. The 6 × His tag is typically attached to the C-terminus or N-terminus of the protein of interest. His tag protein can be specifically combined with various metal ions, such as Ca ²⁺ 、Mg ²⁺ 、Ni ²⁺ 、Cu ²⁺ 、Fe ³⁺ And the like. Due to Ni ²⁺ Some of the special properties of (2), currently loaded with Ni ²⁺ The resin is widely applied to separation and purification of His label recombinant protein. The resin particles are covalently bonded to Ni ²⁺ Combined, commonly known as Ni ²⁺ Is a ligand. Ni ²⁺ Specific interaction with His tag, ni ²⁺ Binding to the His tag is both specific and reversible. Carrying Ni ²⁺ In the process of separating and purifying the protein, the resin is specifically combined with the target protein containing the His label in the recombinant escherichia coli cell disruption protein solution, then the His label target protein is extracted from the protein mixed solution, and then the purified product of the recombinant target protein is obtained through competitive combination of eluent. Ion exchange chromatography is the most common method for protein separation and purification, and can be classified into cation exchange chromatography and anion exchange chromatography. The principle of separating and purifying proteins by the ion exchange method is to separate and purify proteins according to the characteristic of acid-base amphoteric dissociation of the proteins, so the ion exchange method is suitable for separating and purifying various proteins and has wide application. According to the method, protein separation and purification can be carried out under different pH values and different charges, the cation exchange resin is combined with the protein with positive charges, the protein with positive charges is adsorbed on the resin, and then the target protein is eluted by increasing the concentration of an eluent. Anion exchangeThe resin is combined with the protein with negative charge, the protein with negative charge is adsorbed on the resin, and then the target protein is eluted by increasing the salt concentration of the eluent. The preparation technology for protein separation and purification resin is rapidly developed at present, and the resin with the size from dozens of microns to one hundred microns can be prepared in both laboratory and industrial scales. This provides a raw material for the development of new technologies for the separation and purification of proteins based on resin materials.

The solid-liquid mixing method can be various, such as rotary stirring, ultrasonic mixing, heating and mixing. Compared with other mixing modes, the rotary stirring and mixing mode has the following advantages: 1. the rotary stirring and uniform mixing treatment capacity is large, and the method is suitable for treating large-batch samples. 2. The energy consumption required by rotary stirring and uniform mixing is low, and the electric energy is directly converted into mechanical stirring force. The energy consumption of enterprises can be reduced, and a larger profit margin is brought. 3. The operation method of the rotary stirring and blending is simple, and excessive manpower is not required to be consumed. Solid-liquid separation refers to a process of separating a solid-liquid two-phase system from a liquid-solid two-phase system. The solid-liquid separation method includes centrifugal separation and filtration separation. The principle of centrifugal separation is that the separation is carried out according to different densities of various substances and different settling speeds under the action of centrifugal force. The centrifugation method can be classified into differential centrifugation and density gradient centrifugation, depending on the manner of centrifugation. The filtration separation is carried out according to different particle sizes of all phases in a mixed system, and the filtration process is mainly used in the process operation of coal chemical industry and biological medicine.

Disclosure of Invention

Technical problem to be solved

In order to avoid the defects of the prior art, the invention provides a method and a device for efficiently separating and purifying protein based on a uniform mixing-liquid removing process

Technical scheme

A method for efficiently separating and purifying protein based on a uniform mixing-liquid removing process is characterized by comprising the following steps:

step 1: loading large particles with Ni ²⁺ Mixing the resin with 50mmol/L Tris-HCl resin equilibrium liquid at a mass/volume ratio of 1.25%Spin-drying and dehydrating;

step 2: adding colibacillus with labeled target protein in the same volume to ferment and recombine the expression product to make the Ni carried by large particles ²⁺ The resin is specifically combined with the target protein with the label in the fermentation product, and the combination time is 40min; then centrifugal drying is carried out to remove liquid, and the residual protein solution in the fermentation product is subjected to solid-liquid separation to discharge liquid;

and step 3: then adding the solution I for cleaning, spin-drying and dehydrating, and adding the solution II for cleaning, spin-drying and dehydrating to gradually clean the mixed protein.

The solution I: 50mmol/L Tris-HCl,300mmol/L NaCl,50mmol/L imidazole, pH 8.0;

and the solution II: 50mmol/L Tris-HCl,300mmol/L NaCl,100mmol/L imidazole, pH 8.0

And 4, step 4: adding solution III to competitively bind the target protein with the label to elute the target protein from the large-particle Ni2+ resin;

the solution III is as follows: 50mmol/L Tris-HCl,300mmol/L NaCl,500mmol/L imidazole, pH 8.0;

and 5: dialyzing and desalting by a dialysis method to obtain protein;

step 6: the protein is freeze-dried to prepare the high-purity protein.

A device for realizing the high-efficient separation and purification method of protein in the process of mixing and liquid removal is characterized by comprising a fixed bracket 1, a motor 2, an outer cylinder 4 and an inner cylinder 5; the outer cylinder 4 is arranged between the upper frame and the lower frame of the fixed support 1, a capping inner cylinder 5 with a hole at the bottom is arranged in the outer cylinder, the upper part of the inner cylinder 5 is connected with a shaft of the motor 2, and the motor drives the rotation; the upper part of the inner cylinder 5 is provided with two feeding holes 3, a filter cloth cylinder is lined in the cylinder, and the bottom is provided with a liquid outlet hole 6 and a liquid removal switch.

The filter cloth cylinder is 500 meshes.

Advantageous effects

The invention provides a method and a device for efficiently separating and purifying protein based on a uniform mixing-liquid removal process ²⁺ The resin binds specifically to the protein of interest in the protein solution to be purified. First of all carryNi ²⁺ The resin is fully combined with the target protein, and then the solid-liquid separation of the resin combined with the target protein and the residual protein liquid after purification is realized through the rotation of the filter cloth and the inner cylinder of the device. And cleaning with a gradient-concentration mixed protein cleaning solution, finally adding an elution solution to collect the target protein, and dialyzing, freezing and drying to prepare the high-purity target protein.

Compared with the traditional protein chromatography, the method has the following advantages:

1. compared with the traditional chromatographic separation and purification method, the method for efficiently separating the protein by non-chromatography does not need professional technicians to complete the filling of the protein purification medium, so the labor cost can be saved to a certain extent. In the face of the market with large protein demand, the chromatographic protein separation and purification needs high cost, and the invention has the advantage of low cost. The invention has great investment application prospect. If the invention is applied to the industrialized protein separation and purification process, the production cost of the protein can be greatly reduced.

2. At present, a large amount of target protein can be expressed by fermentation through constructing a recombinant prokaryotic expression system. However, chromatography is currently not capable of handling high titer recombinant protein products, and the device can handle large batches of protein products by only combining and amplifying the various components. The invention can overcome the bottleneck that the chromatography technology can not process the high-titer recombinant protein product.

3. The traditional chromatography method needs 5-6 h for separating and purifying protein, and the invention can shorten the time to 30min. The invention can shorten the time for separating and purifying the protein, thereby improving the protein purification efficiency.

Drawings

FIG. 1: device appearance diagram

FIG. 2: internal structure diagram of device

1- - -a fixed bracket 2- - -a motor 3- - -a sample inlet 4- - -an outer cylinder 5- - -an inner cylinder 6- - -a sample outlet

Detailed Description

The invention will now be further described with reference to the following examples, and the accompanying drawings:

the whole device consists of the following structures: 1. a speed-controllable motor 2.2. A rotatable capping inner cylinder 5 with a hole at the bottom, and two feeding holes 3 are arranged at the upper part of the inner cylinder 5. 3. An opening is used for fixing the outer cylinder 4, and the bottom of the outer cylinder 4 is provided with a liquid outlet hole. 4. The lining of the inner cylinder 5 is a 500-mesh filter cloth cylinder. The working principle of the device is that the motor 2 drives the inner cylinder 5 to rotate to generate centrifugal force to carry out solid-liquid separation of the nickel-loaded resin and the protein purification solution, so that the low-cost and high-efficiency preparation of the protein is achieved. As shown in the drawings, fig. 1 is an external view of the apparatus, and fig. 2 is an internal configuration view of the apparatus.

The efficient protein separating and purifying method based on the uniform mixing-liquid removing process comprises the following steps:

(1) 6g of large particles loaded with Ni ²⁺ The resin is filled into the filter cloth lining of the device, 70mL of 50mmol/L Tris-HCl equilibrium resin is added, and the solution is removed after the resin equilibrium is finished.

(2) The inner cylinder of the device is rotated to carry Ni on large particles ²⁺ The resin is specifically combined with protein containing a label in 70mL of recombinant escherichia coli expression intracellular product release protein liquid, and the combination time is 25min.

(3) And (3) after the step 2 is finished, filtering the protein solution by a filter cloth cylinder of the device and performing centrifugal drying on the inner cylinder of the device to perform solid-liquid separation on the labeled resin combined with the target protein and the residual protein solution in the supernatant of the cell disruption, and discharging the liquid.

(4) And gradually cleaning the hybrid protein by using 120-140 mL of solution I50 mmol/L Tris-HCl,300mmol/L NaCl,50mmol/L imidazole, pH 8.0, 120140 mL of solution II 50mmol/L Tris-HCl,300mmol/L NaCl,100mmol/L imidazole and pH 8.0.

(5) After the step 4 is finished, the target protein is eluted from the eluted large-particle resin by using 60-70 mL of solution III 50mmol/L Tris-HCl,300mmol/L NaCl,500mmol/L imidazole and pH 8.0 for competitive binding with the target protein.

(6) Because the purified protein sample has high salt concentration, the specification of a dialysis bag, 3500D, is required for dialysis and desalination.

7 freeze-drying the protein of step 6 to prepare the high-purity protein.

Example (b):

large particle polyacrylic acid loaded Ni ²⁺ Extracting His-tag SOD with resin.

The specific process comprises the following steps:

1) Weighing 3g of large-particle polyacrylic acid Ni ²⁺ The resin is placed in a filter cloth liner of the device, 70mL of 50mmol/L Tris-HCl buffer solution is added into an inner cylinder of the device, and a motor is started to drive the inner cylinder to rotate to balance the resin.

2) And (3) after the step 1 is finished, turning on a liquid removal switch, and throwing out liquid by utilizing the rotation of the inner cylinder.

3) 70mL of His label SOD cell disruption supernatant is added into the inner cylinder of the device, and a motor is turned on to drive the inner cylinder to rotate. Make Ni carry ²⁺ The resin was mixed well with the cell disruption supernatant. And after the combination is finished, the liquid removal switch is turned on, and the combination residual liquid is thrown out by utilizing the rotation of the inner cylinder.

4) Adding 70mL of solution I into the inner cylinder of the device, and cleaning the solution I attached to the Ni load ²⁺ Other hetero-proteins on the surface of the resin. After the cleaning is finished, the liquid removal switch is turned on, and the cleaning liquid is thrown out by utilizing the rotation of the inner cylinder.

5) And (4) repeating the step.

6) 70mL of solution II is added into the inner cylinder of the device, and the Ni-loaded particles are cleaned and attached to the inner cylinder ²⁺ Remaining heteroproteins on the resin surface. After the cleaning is finished, the liquid removal switch is turned on, and the cleaning liquid is thrown out by utilizing the rotation of the inner cylinder.

7) And 6, repeating the step.

8) Adding 70mL of solution III into the inner cylinder of the device, and competitively eluting the loaded Ni ²⁺ Resin-bound His-tag SOD of a target protein. After the elution is finished, a liquid removal switch is turned on, and the SOD containing the His label is thrown out by utilizing the rotation of the inner cylinder.

9) Dialyzing with 3500D dialysis bag for 48h to obtain desalted His tag-containing SOD.

10 drying with vacuum freeze dryer to obtain powder of His-tagged SOD.

Example (b):

the egg white lysozyme is extracted from the large-particle LSR-7B ion exchange resin.

The specific process comprises the following steps:

resin pretreatment:

1) Weighing 10g of large-particle LSR-7B ion exchange resin, placing the resin in the experimental device of the invention, and soaking the resin for 12 hours by using 1mol/L hydrochloric acid.

2) Abandoning the supernatant, and turning on a motor to drive the inner cylinder to rotate and throw out the waste liquid. Washing with distilled water until pH is about 6.0.

3) Soaking the resin in 80 deg.C hot water for 4 hr, filtering, and turning on motor to drive the inner cylinder to rotate and throw out waste liquid.

4) Soaking the resin in 1mol/L sodium hydroxide solution for 12h, washing with distilled water until the pH is about 9.0, and turning on a motor to drive an inner cylinder to rotate and throw out waste liquid.

Egg white pretreatment:

5, taking egg white, diluting with 100mmol/L NaCl solution to obtain a solution with the concentration of 1:1 diluting egg white, and filtering with gauze to remove impurities.

6 mixing the pretreated egg white and the pretreated resin in the experimental device in equal volume, and rotating, stirring and adsorbing the inner cylinder of the experimental device for 6 hours. After the adsorption is finished, residual egg white liquid is collected, and the motor is started to drive the inner cylinder to rotate and throw out waste liquid.

7) Adding 10% ammonium sulfate with the same volume into the device, stirring and eluting for 1h, taking the supernatant after the elution is finished, and turning on a motor to drive an inner cylinder to rotate and throw out waste liquid.

8) The lysozyme is prepared by dialysis and freeze drying.

Claims (2)

1. The utility model provides a realize mixing-high-efficient separation and purification's of protein device of liquid process that takes off, the device's major structure includes fixed bolster (1), motor (2), introduction port (3), urceolus (4), inner tube (5), appearance mouth (6) and takes off the liquid switch, its characterized in that as follows:

the fixing support (1) is composed of two parts, the upper part of the fixing support (1) is connected with the motor (2) through a bearing, so that the motor (2) is fixed on the upper part of the fixing support (1) to play a role in fixing the motor (2) and the inner cylinder (5), and the lower part of the fixing support (1) plays a role in supporting and fixing the outer cylinder (4);

the motor (2) provides a power source for the whole device, and the motor (2) is directly connected with the inner cylinder (5);

the sample inlet (3) is a sample inlet for cell disruption supernatant in the whole device to enter the device for separation and purification; in the process of protein separation and purification, all cleaning solution and eluent enter the device through the sample inlet (3), and the cleaning solution in the device also enters the device through the sample inlet (3);

the outer cylinder (4) is a liquid collecting device arranged outside the inner cylinder (5), the bottom of the outer cylinder is provided with a sample outlet (6) and a liquid removal switch, and when liquid flows out of the inner cylinder (5), the liquid is collected by the outer cylinder (4) and flows out through the sample outlet (6); the upper part of the outer cylinder (4) is open so as to facilitate the loading and unloading of the inner cylinder (5), and the bottom of the outer cylinder is provided with a hole so as to facilitate the liquid to flow out;

the upper portion of inner tube (5) is equipped with two introduction ports (3), has a filter cloth section of thick bamboo in the section of thick bamboo, and inner tube (5) realize autogyration by motor (2) drive to can realize the variable speed autogyration of inner tube (5) through motor (2).

2. A method for efficiently separating and purifying protein by using the device of claim 1 to carry out a blending-liquid removal process is characterized by comprising the following steps:

step 1: loading large particles with Ni ²⁺ After the resin is filled into a filter cloth lining, mixing the resin with 50mmol/L Tris-HCl resin equilibrium liquid according to the mass-to-volume ratio of 1.25 percent, carrying out equilibrium reaction for 5min to obtain the pretreated resin, and centrifugally spin-drying and dehydrating after the resin is balanced;

step 2: adding an equal volume of the recombinant expression product of E.coli with the tagged target protein, and rotating the inner cylinder (5) in the device of claim 1 to make the large particles Ni-loaded ²⁺ The resin is specifically combined with the target protein with the label in the fermentation product, and the combination time is 40min; then centrifugal drying is carried out to remove liquid, and the residual protein solution in the fermentation product is subjected to solid-liquid separation to discharge liquid;

and step 3: then adding the solution I for cleaning, spin-drying and dehydrating, and adding the solution II for cleaning, spin-drying and dehydrating to gradually clean the mixed protein;

the solution I: 50mmol/L Tris-HCl,300mmol/L NaCl,50mmol/L imidazole, pH 8.0;

and the solution II: 50mmol/L Tris-HCl,300mmol/L NaCl,100mmol/L imidazole, pH 8.0;

and 4, step 4: adding solution III to competitively bind the target protein with the label to elute the target protein from the large-particle Ni2+ resin, and then utilizing an inner cylinder (5) to spin and throw out the target protein;

the solution III is as follows: 50mmol/L Tris-HCl,300mmol/L NaCl,500mmol/L imidazole, pH 8.0;

and 5: dialyzing and desalting by a dialysis method to obtain protein;

step 6: freeze-drying the protein to prepare high-purity protein;

wherein the step 1-3 of spin-drying and liquid-removing is carried out by the rotation of the inner drum (5) in the device of claim 1.

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