Large-scale inclusion body protein dialysis renaturation device

Abstract

The utility model provides a large-scale inclusion body protein dialysis renaturation device, and relates to the technical field of protein dialysis renaturation. The inclusion body protein dialysis renaturation device comprises a renaturation liquid cavity, a dialysis membrane, a protein liquid cavity, a first renaturation liquid container, a second renaturation liquid container and a binary gradient pump; the volumes of the renaturation liquid cavity, the protein liquid cavity, the first renaturation liquid container and the second renaturation liquid container are relatively large, so that the treatment capacity of the device is large; the binary gradient pump is used for presetting the solution proportion for mixing, so that the operation process is simple and convenient, meanwhile, the conditions of the dialysis process are met, and the protein renaturation efficiency is improved. The problems that in the prior art, the treatment capacity is small, operation is tedious, the conditions of the dialysis process are difficult to control, and the cost is increased due to the fact that the use amount of the solution is greatly increased when dialysis external liquid is continuously replaced are solved.

Description

Technical Field

[0001] The utility model relates to the technical field of protein dialysis refolding, in particular to a large-scale inclusion body protein dialysis refolding device. Background Art

[0002] Using E. coli prokaryotes to express target proteins is one of the means of large-scale industrial preparation of recombinant proteins. Its advantages are that it can obtain gene expression products in a relatively short period of time, and the cost required is relatively low, the operation method is relatively simple, and the number of proteins that can be expressed is also relatively large. However, due to the excessively fast protein expression rate and the lack of late protein modification, the protein will cause abnormal folding inside the E. coli, exposing the hydrophobic core to the outside, thereby forming insoluble inclusion body proteins. In order to obtain a protein with the correct structure, soluble and active, it is necessary to use a denaturant such as a high concentration of guanidine hydrochloride or urea solution to denature and dissolve the insoluble inclusion body protein, and then reduce the concentration of the denaturant in the protein solution by dialysis or dilution, so that the denatured protein slowly forms the correct spatial folding, and finally becomes a water-soluble and biologically active target protein.

[0003] The basic structure of traditional devices is to place the protein solution to be renatured in a dialysis bag, then immerse the dialysis bag in a dialysis external fluid, and continuously replace the dialysis external fluid to reduce the concentration of guanidine hydrochloride or urea in the dialysis external fluid, thereby reducing the concentration of the denaturant in the protein solution and achieving protein renaturation. However, since protein renaturation has certain requirements for the protein concentration in the protein solution, failure to meet the requirements will cause a large amount of protein precipitation, resulting in renaturation failure; in addition, traditional dialysis renaturation methods also have the problems of small processing capacity, cumbersome operation, and difficult to control the conditions of the dialysis process. Continuously replacing the dialysis external fluid will greatly increase the amount of solution used, resulting in increased costs, and other problems. Utility Model Content

[0004] Therefore, the technical problem to be solved by the utility model is to overcome the defects of the prior art such as small processing volume, complicated operation, difficult to control the conditions of the dialysis process, and continuous replacement of the dialysis external fluid which will greatly increase the amount of solution used and lead to increased costs, thereby providing a large-scale inclusion body protein dialysis renaturation device.

[0005] A large-scale inclusion body protein dialysis refolding device comprises a dialysis device and a fluid replacement device;

[0006] The dialysis device comprises a refolding liquid cavity, a dialysis membrane, a protein liquid cavity, a refolding liquid outlet and a refolding liquid inlet; one side of the dialysis membrane is connected to the refolding liquid cavity, and the other side of the dialysis membrane is connected to the protein liquid cavity;

[0007] The liquid replacement device comprises a first refolding liquid container, a second refolding liquid container and a power device, the first refolding liquid container is connected to the power device via a first hose, and the second refolding liquid container is connected to the power device via a second hose;

[0008] The surface of the refolding liquid cavity is provided with a refolding liquid outlet and a refolding liquid inlet, and the refolding liquid outlet is arranged above the refolding liquid inlet;

[0009] The power device is connected to the refolding liquid inlet through a third hose, and the second refolding liquid container is connected to the refolding liquid outlet through a hard tube.

[0010] Furthermore, one side of the dialysis membrane is connected to the refolding liquid cavity via a sealing strip, and the other side of the dialysis membrane is connected to the protein liquid cavity via a sealing strip.

[0011] Furthermore, the first refolding liquid container is a refolding liquid container without a denaturant, and the second refolding liquid container is a refolding liquid container containing a denaturant.

[0012] Furthermore, the power device is a binary gradient pump.

[0013] Furthermore, the volumes of the refolding liquid chamber and the protein liquid chamber are both 250 ml.

[0014] Furthermore, the volumes of the first refolding liquid container and the second refolding liquid container are both 2 liters.

[0015] The beneficial effects of the utility model are:

[0016] The large-scale inclusion body protein dialysis refolding device provided by the utility model uses different hoses and hard tubes to transport solutions between the refolding liquid cavity, the protein liquid cavity, the first refolding liquid container and the second refolding liquid container, thereby reducing the concentration of the denaturant, so that the protein solution containing the denaturant gradually becomes a water-soluble and biologically active target protein, reducing the use of various solution amounts, and also reducing costs. In the technical solution provided by the utility model, the volume of the refolding liquid cavity, the protein liquid cavity, the first refolding liquid container and the second refolding liquid container is large, and the protein dialysis process has a large processing capacity. In the technical solution provided by the utility model, a dialysis membrane is used between the refolding liquid cavity and the protein liquid cavity to reduce the concentration of the denaturant, and a binary gradient pump is used to pre-set the solution ratio and mix, so that the operation process is simple and the conditions of the dialysis process are also met. In the technical solution provided by the utility model, the device can be expanded by connecting multiple groups of dialysis devices in parallel with a liquid exchange device. The refolding of multiple proteins is achieved at the same time, and production efficiency is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the utility model or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the utility model. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 It is a schematic diagram of the overall structure of the utility model;

[0019] Figure 2 It is a partial structural expansion diagram of the dialysis device of the utility model;

[0020] Figure 3 It is a partial plan view of the liquid changing device of the utility model.

[0021] Description of reference numerals:

[0022] 1-renaturation liquid chamber; 2-dialysis membrane; 3-protein liquid chamber;

[0023] 4-renaturation liquid outlet; 5-renaturation liquid inlet; 6-sealing strip;

[0024] 7-first refolding liquid container; 8-second refolding liquid container; 9-power equipment;

[0025] 101-first hose; 102-second hose; 103-third hose;

[0026] 104-Hard pipe. DETAILED DESCRIPTION

[0027] The technical solution of the utility model will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the utility model, not all of the embodiments. Based on the embodiments of the utility model, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the utility model.

[0028] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0029] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installation", "connection" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0030] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0031] like Figure 1 , Figure 2 and Figure 3 The inclusion body protein dialysis refolding device shown includes: a dialysis device and a fluid exchange device; the dialysis device includes a refolding liquid chamber 1, a dialysis membrane 2, a protein liquid chamber 3, a refolding liquid outlet 4 and a refolding liquid inlet 5; one side of the dialysis membrane 2 is connected to the refolding liquid chamber 1, and the other side of the dialysis membrane 2 is connected to the protein liquid chamber 3; the fluid exchange device includes a first refolding liquid container 7, a second refolding liquid container 8 and a power device 9, the first refolding liquid container 7 is connected to the power device 9 through a first hose 101, and the second refolding liquid container 8 is connected to the power device 9 through a second hose 102; the surface of the refolding liquid chamber 1 is provided with a refolding liquid outlet 4 and a refolding liquid inlet 5, and the refolding liquid outlet 4 is arranged above the refolding liquid inlet 5; the power device 9 is connected to the refolding liquid inlet 5 through a third hose 103, and the second refolding liquid container 8 is connected to the refolding liquid outlet 4 through a hard tube 104.

[0032] The inclusion body protein dialysis refolding device reduces the concentration of the denaturant by transferring the solution between the refolding liquid chamber 1, the protein liquid chamber 3, the first refolding liquid container 7 and the second refolding liquid container 8, so that the protein solution containing the denaturant gradually becomes a water-soluble and biologically active target protein, reduces the use of various solutions, has a large processing volume, and also reduces costs and simplifies the operation process. The power device 9 can be used to pre-set the mixing ratio of the solution, which simplifies the operation process while also meeting the conditions of the entire dialysis process.

[0033] like Figure 1 , Figure 2 and Figure 3 As shown, in this embodiment, the refolding liquid chamber 1, the dialysis membrane 2 and the protein liquid chamber 3 are arranged according to Figure 2 As shown in the installation, one side of the dialysis membrane 2 is connected to the refolding liquid chamber 1 through a sealing strip 6, and the other side of the dialysis membrane 2 is connected to the protein liquid chamber 3 through a sealing strip 6. The dialysis membrane 2 is installed between the two liquid chambers using the sealing strip 6, which is beneficial to completely isolate the two liquid chambers and prevent leakage.

[0034] like Figure 1 , Figure 2 and Figure 3 As shown, in this embodiment, the first refolding liquid container 7 is a refolding liquid container without a denaturant, and the second refolding liquid container 8 is a refolding liquid container containing a denaturant; the power device 9 is a binary gradient pump; the two refolding liquid containers are beneficial to the protein dialysis refolding process and accelerate the protein refolding speed; the binary gradient pump can pre-set the mixing ratio of the solution, simplifying the protein dialysis refolding process and simple operation.

[0035] like Figure 1 , Figure 2 and Figure 3 As shown, in this embodiment, the refolding liquid outlet 4 is arranged above the refolding liquid inlet 5; it is beneficial for the solution to enter the refolding liquid chamber 1 through the refolding liquid inlet 5 from the binary gradient pump, and the solution to enter the second refolding liquid container 8 through the refolding liquid outlet 4. The volumes of the refolding liquid chamber 1 and the protein liquid chamber 3 are both 250 ml; the volumes of the first refolding liquid container 7 and the second refolding liquid container 8 are both 2 liters; a larger container volume will enable the device to achieve large-scale protein dialysis refolding.

[0036] like Figure 1 , Figure 2 and Figure 3 As shown, in this embodiment, the inclusion body protein is dissolved with a denaturant solution to prepare a protein solution, the concentration is adjusted to 100 μg / mL, and poured into the protein liquid chamber 3, the refolding solution containing a high concentration of denaturant is poured into the refolding liquid chamber 1 and the second refolding liquid container 8, and the refolding solution without a denaturant is poured into the first refolding liquid container 7; then a binary gradient pump is used to mix the solutions in the first refolding liquid container 7 and the second refolding liquid container 8 at a set ratio, and after mixing, the solution is transported to the refolding liquid chamber 1 through the refolding liquid inlet 5 through the third hose 103, and when the liquid level in the refolding liquid chamber 1 reaches the refolding liquid outlet 4, the solution in the refolding liquid chamber 1 will flow back to the second refolding liquid container 8 through the communicating vessel principle. Since the proportion of the solutions is set by using a binary gradient pump, the proportion of the refolding liquid in the second refolding liquid container 8 gradually decreases, and the proportion of the refolding liquid in the first refolding liquid container 7 gradually increases; thereby, the denaturant concentrations in the refolding liquid chamber 1 and the second refolding liquid container 8 gradually decrease until the denaturant concentration in the refolding liquid chamber 1 becomes zero. Since there is a dialysis membrane 2 (semi-permeable membrane) between the refolding liquid chamber 1 and the protein liquid chamber 3, the denaturant concentration in the protein liquid chamber 3 also becomes zero, and the protein refolding is completed.

[0037] like Figure 1 , Figure 2 and Figure 3As shown, in this embodiment, the EGF inclusion bodies were dissolved with a protein dissolving solution (50 mM Tris-HCl solution containing 8 M urea, pH 8.0) and the concentration was adjusted to 100 μg / mL. 200 mL of the diluted protein solution was poured into the protein liquid chamber 3, and 200 mL of the denaturing solution containing a denaturant (50 mM Tris-HCl solution containing 8 M urea, 5 mM oxidized glutathione, 10 mM reduced glutathione, pH 8.0) was poured into the refolding liquid chamber 1. Pour 2L of refolding solution without denaturant (50 mM Tris-HCl solution containing 8M urea, 5 mM oxidized glutathione, 10 mM reduced glutathione, pH 8.0) into the first refolding solution container 7, and pour 1L of refolding solution containing denaturant (50 mM Tris-HCl solution containing 8M urea, 5 mM oxidized glutathione, 10 mM reduced glutathione, pH 8.0) into the second refolding solution container 8. Turn on the binary gradient pump, set the liquid ratio in the second refolding solution container 8 to gradually decrease (100%-0%), and the liquid ratio in the first refolding solution container 7 to gradually increase (0%-100%), and the duration is 48 hours. Then, according to the liquid ratio of the first refolding solution container 7 to 100%, run for 24 hours until the liquid in the first refolding solution container 7 is exhausted. Collect the protein liquid from the protein liquid chamber 3.

[0038] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention of the utility model.

Claims

1. A large-scale inclusion body protein dialysis renaturation device, characterized in that: The inclusion body protein dialysis and refolding device comprises: a dialysis device and a fluid replacement device; The dialysis device comprises a refolding liquid chamber (1), a dialysis membrane (2), a protein liquid chamber (3), a refolding liquid outlet (4) and a refolding liquid inlet (5); one side of the dialysis membrane (2) is connected to the refolding liquid chamber (1), and the other side of the dialysis membrane (2) is connected to the protein liquid chamber (3); The liquid replacement device comprises a first refolding liquid container (7), a second refolding liquid container (8) and a power device (9); the first refolding liquid container (7) is connected to the power device (9) via a first hose (101), and the second refolding liquid container (8) is connected to the power device (9) via a second hose (102); The surface of the refolding liquid chamber (1) is provided with a refolding liquid outlet (4) and a refolding liquid inlet (5), and the refolding liquid outlet (4) is arranged above the refolding liquid inlet (5); The power device (9) is connected to the refolding liquid inlet (5) via a third hose (103), and the second refolding liquid container (8) is connected to the refolding liquid outlet (4) via a hard tube (104).

2. A large-scale inclusion body protein dialysis renaturation device according to claim 1, characterized in that: One side of the dialysis membrane (2) is connected to the refolding liquid chamber (1) via a sealing tape (6), and the other side of the dialysis membrane (2) is connected to the protein liquid chamber (3) via a sealing tape (6).

3. A large-scale inclusion body protein dialysis renaturation device according to claim 1, characterized in that: The first refolding liquid container (7) is a refolding liquid container that does not contain a denaturing agent, and the second refolding liquid container (8) is a refolding liquid container that contains a denaturing agent.

4. A large-scale inclusion body protein dialysis renaturation device according to claim 1, characterized in that: The power device (9) is a binary gradient pump.

5. A large-scale inclusion body protein dialysis renaturation device according to claim 1, characterized in that: The volumes of the refolding liquid chamber (1) and the protein liquid chamber (3) are both 250 ml.

6. A large-scale inclusion body protein dialysis renaturation device according to claim 1, characterized in that: The volumes of the first refolding liquid container (7) and the second refolding liquid container (8) are both 2 liters.

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