FULLY-AUTOMATIC PROTEIN PURIFICATION SYSTEM DEVICE AND USE THEREOF

Abstract

A fully-automatic protein purification system device includes a chromatography unit, a first drive unit, a connecting pipeline, a locating unit, a second drive unit, a first container, a second container, a first valve, a second valve, and a control unit. The fully-automatic protein purification system device can fully automate the protein chromatography purification with a simple device structure and a low cost, has low requirements for the quality of a sample solution, will not cause blockage of a pipeline, has a wide application range, can greatly improve the automation of protein chromatography purification in the biology field, and can reduce the manual investment.

Claims

1. A fully-automatic protein purification system device, comprising a chromatography unit, a first drive unit, a connecting pipeline, a locating unit, a second drive unit, a first container, a second container, a first valve, a second valve, and a control unit, wherein the connecting pipeline has a first end connected to the chromatography unit and a second end connected to the locating unit; the second drive unit drives the locating unit; the first container is connected to an upper part of the chromatography unit with a first pipeline through a first two-way valve, and the second container is connected to the upper part of the chromatography unit with a second pipeline through a second two-way valve; the first drive unit drives a solution in the first container to flow through the chromatography unit, and a liquid flowing out is collected in the second container located below the connecting pipeline through the connecting pipeline; the connecting pipeline rotates with the locating unit, such that an outlet of the connecting pipeline is located above the second container.

2. The fully-automatic protein purification system device according to claim 1, wherein the chromatography unit comprises a liquid level detector.

3. The fully-automatic protein purification system device according to claim 1, wherein the chromatography unit is a chromatography column.

4. The fully-automatic protein purification system device according to claim 1, wherein the connecting pipeline is a hose.

5. The fully-automatic protein purification system device according to claim 1, wherein the locating unit is a locating column; and the locating unit is configured to rotate 360°.

6. The fully-automatic protein purification system device according to claim 1, wherein the second drive unit is a stepper motor.

7. The fully-automatic protein purification system device according to claim 1, further comprising: a third container, a fourth container, a fifth container, a sixth container.

8. The fully-automatic protein purification system device according to claim 1, further comprising: a third container, a fourth container, a fifth container, a sixth container, a third valve, a fourth valve, a fifth valve, and a sixth valve, wherein the third container, the fourth container, the fifth container, and the sixth container are connected to the upper part of the chromatography unit with third pipelines through the third valve, the fourth valve, the fifth valve, and the sixth valve, respectively; the third valve is a third two-way valve; the fourth valve is a fourth two-way valve; the fifth valve is a fifth two-way valve; the sixth valve is a sixth two-way valve; and each of the first two-way valve, the second two-way valve, the third two-way valve, the fourth two-way valve, the fifth two-way valve, and the sixth two-way valve is a two-way solenoid valve.

9. The fully-automatic protein purification system device according to claim 1, wherein the first valve and the second valve are normally-closed valves and are opened only after being powered up; and at any time point, only one valve of the first valve and the second valve is opened and the remaining one of the first valve and the second valve is closed.

10. A method of use of the fully-automatic protein purification system device according to claim 1 in a purification of a protein.

11. The fully-automatic protein purification system device according to claim 1, wherein the first drive unit is a peristaltic pump.

12. The fully-automatic protein purification system device according to claim 1, wherein the the second drive unit is a motor.

13. The fully-automatic protein purification system device according to claim 1, wherein the the first valve is the first two-way valve.

14. The fully-automatic protein purification system device according to claim 1, wherein the the second valve is the second two-way valve.

15. The fully-automatic protein purification system device according to claim 2, wherein the the chromatography unit is a chromatography column.

16. The fully-automatic protein purification system device according to claim 2, wherein the connecting pipeline is a hose.

17. The fully-automatic protein purification system device according to claim 3, wherein the connecting pipeline is a hose.

18. The fully-automatic protein purification system device according to claim 4, wherein the horse is a silicone hose.

19. The fully-automatic protein purification system device according to claim 3, wherein the locating unit is a locating column; and the locating unit is configured to rotate 360°.

20. The fully-automatic protein purification system device according to claim 4, wherein the locating unit is a locating column; and the locating unit is configured to rotate 360°.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is a schematic structural diagram of the fully-automatic protein purification system device of the present disclosure.

[0035] FIG. 2 is a schematic diagram illustrating the working status of the fully-automatic protein purification system device of the present disclosure, where a silicone hose 2 is located directly above a second container 6.

REFERENCE NUMERALS

[0036] 0 represents a chromatography column; 1 represents a peristaltic pump; 2 represents a silicone hose; 3 represents a locating column; 4 represents a stepper motor; 5 represents a first container; 6 represents a second container; 7 represents a third container; 8 represents a fourth container; 9 represents a fifth container; 10 represents a sixth container; 51 represents a first two-way solenoid valve; 61 represents a second two-way solenoid valve; 71 represents a third two-way solenoid valve; 81 represents a fourth two-way solenoid valve; 91 represents a fifth two-way solenoid valve; 101 represents a sixth two-way solenoid valve; and 11 represents a liquid level detector.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present disclosure is further described below in conjunction with specific examples, and these examples are implemented under the premise of the technical solutions of the present disclosure. It should be understood that these examples are provided merely to illustrate the present disclosure rather than to limit the scope of the present disclosure.

EXAMPLE 1

A Fully-Automatic Protein Purification System Device

[0038] FIG. 1 shows the fully-automatic protein purification system device. The fully-automatic protein purification system device includes a chromatography column 0, a peristaltic pump 1, a silicone hose 2, a locating column 3, a stepper motor 4, a first container 5, a second container 6, a third container 7, a fourth container 8, a fifth container 9, a sixth container 10, a first two-way solenoid valve 51, a second two-way solenoid valve 61, a third two-way solenoid valve 71, a fourth two-way solenoid valve 81, a fifth two-way solenoid valve 91, a sixth two-way solenoid valve 101, a liquid level detector 11, and a circuit board control system thereof. The stepper motor 4 drives the locating column 3 connected thereto, such that the locating column can rotate 360° . The silicone hose 2 has one end connected to an outlet of the chromatography column 0, a middle part clamped on the peristaltic pump, and the other end fixed on a crossbar of the locating column. According to a set instruction, the silicone hose 2 rotates with the locating column 3, such that an outlet of the silicone hose 2 may be located above any one of the first container 5, the second container 6, the third container 7, the fourth container 8, the fifth container 9, and the sixth container 10 (the outlet is located above the third container 7 in FIG. 1).

[0039] The chromatography column 0 is divided into an upper part and a lower part, where the upper part of the chromatography column 0 communicates with a container through a pipeline, and the lower part of the chromatography column 0 (the grid part) is filled with an affinity medium. An outlet connecting pipeline is provided at the bottom of each of the first container 5, the second container 6, the third container 7, the fourth container 8, the fifth container 9, and the sixth container 10. The connecting pipeline is accordingly first connected to the first two-way solenoid valve 51, the second two-way solenoid valve 61, the third two-way solenoid valve 71, the fourth two-way solenoid valve 81, the fifth two-way solenoid valve 91, or the sixth two-way solenoid valve 101 and then connected to an upper interface of the chromatography column 0 through a pipeline. All solenoid valves are normally-closed solenoid valves and are opened only after being powered up. According to a preset instruction of an operation program, at any time point, only one solenoid valve is opened and the remaining ones are closed. A lower outlet of the chromatography column 0 is connected to the silicone hose 2, and the silicone hose 2 is controlled and powered by the peristaltic pump 1. According to a preset instruction, a solenoid valve in one of the first container 5, the second container 6, the third container 7, the fourth container 8, the fifth container 9, and the sixth container 10 is opened, the peristaltic pump drives a solution in a corresponding container to flow through the chromatography column 0, and a liquid flowing out is collected in a container located directly below the silicone hose 2 through the silicone hose 2.

[0040] A liquid level detector 11 is provided at an upper position with a specified height inside the chromatography column 0, which is configured to remind a liquid level through the conductivity of a solution. When a liquid flows normally, an AB metal probe above the liquid level detector (as shown in “+” and “−” below 11 in FIG. 1) is immersed in a sample solution, and due to the conductivity of the liquid, the detector will give a high electrical level signal, indicating that the solution is currently flowing normally. When a sample is about to be exhausted, a liquid level during chromatography slowly drops, and at a specified time point, the detector is exposed above the liquid level, and two electrodes on the probe are disconnected, which will give the system a low electrical level signal, indicating that the current liquid is about to be exhausted and the program is about to enter the next stage.

EXAMPLE 2

Control Parameter D esign

[0041] The following three operating parameters can be mainly set for the device of the present disclosure: binding times N, delay time T (s), and liquid flow rate. N represents the number of times a sample solution flows through a chromatography column, which is actually the number of times a medium adsorbs a sample. During manual protein chromatography purification, it is usually enough to conduct resin binding 2 times, and thus N is directly set to 2. The delay time T (s) means that, during an operation of the device, when the electrodes on the AB metal probe of the liquid level detector are just exposed above the liquid level and the system will receive a signal for switching from a high electrical level to a low electrical level, it actually still takes T (s) for the liquid level inside the chromatography column 0 in FIG. 1 to drop to an upper position of an adsorption medium (indicating that the sample is exhausted), and then the next operation is implemented, such as solenoid valve switching. The delay time T needs to be set according to specific parameters of the device, which is related to the liquid flow rate, the inner diameter of the chromatography column, and the height of the liquid level detector. The setting value of the delay time T is determined according to measured experimental results. The liquid flow rate is expressed as an amount (mL) of a liquid flowing through per minute and is related to the rotational speed of the peristaltic pump 1 and the size of the corresponding hose 2, which can be set according to the needs of a user and can also be determined according to the actual measurement results.

EXAMPLE 3

Protein affinity adsorption

[0042] Escherichia coli (E. coli) cells carrying an affinity purification tag protein His*6 were subjected to ultrasonic disruption and then centrifuged at 16,000 g for 30 min to obtain 50 mL of a supernatant, and the supernatant was placed in the first container 5 in FIG. 2. The binding times N was set to 2, the flow rate was set to 1.5 mL/min, and the delay time T was equal to 30 s. A chromatography column filled with a nickel medium was well equilibrated with a 50 mM Tris protein buffer (pH=8) in advance. After the “Adsorption” mode was selected and the automatic program was started, the first two-way solenoid valve 51 connected to the first container 5 was powered up and was allowed to communicate with a pipeline, and the silicone hose 2 was located directly above the second container 6. The peristaltic pump was started to drive a fresh protein solution in the first container 5 to continuously flow through the solenoid valve 51 and then flow through the chromatography column 0 with an affinity medium, and a liquid flowing out after the adsorption was collected in the second container 6 through the silicone hose 2. The liquid level detector 11 was always in a high electrical level status throughout the adsorption process. At about 29 min, the sample solution was about to be exhausted and the liquid level detector was switched to a low electrical level; 30 s later, the system closed the first two-way solenoid valve 51 and opened the second two-way solenoid valve 61, and then the locating column was rotated such that the silicone hose 2 fixed on the locating column was located directly above the first container 5 to collect a sample solution obtained after the second resin binding in the next step. The second resin binding of the protein sample solution was then started, and within a few seconds after the second two-way solenoid valve 61 was opened, the liquid level detector was immersed in the sample solution once again, and the system was switched to a high electrical level. About 30 min later, the liquid level detector was exposed above the liquid level once again and the high electrical level was switched to a low electrical level, such that the second adsorption operation of the protein sample solution was completed, and then all solenoid valves and the peristaltic pump were closed. The entire process was fully automated without human intervention.

EXAMPLE 4

Protein Affinity Adsorption, Impurity Removal, and Elution

[0043] A resin binding operation of a protein sample was exactly the same as Example 2 (the binding times N was set to 2, the flow rate was set to 1.5 mL/min, and the delay time T was equal to 30 s), and the “purification” mode was selected. After the resin binding was conducted twice, the non-specifically-adsorbed other protein was first washed off with 100 mL of a washing buffer (50 mM Tris buffer, 20 mM imidazole, pH=8) placed in the third container 7, the target protein was eluted with 50 mL of an elution buffer (50 mM Tris buffer, 300 mM imidazole, pH=8) placed in the fourth container 8, and other settings were the same as Example 1.

[0044] When the second resin binding was about to be completed (as shown in Example 2), the liquid level detector was switched from a high electrical level to a low electrical level; 30 s later, the system closed the second two-way solenoid valve 61 and opened the third two-way solenoid valve 71, and the silicone hose 2 was driven by the stepper motor 4 such that the silicone hose 2 was located directly above the sixth container 10 to collect a waste liquid resulting from the washing of the chromatography column. The automation of washing-off of the other protein on the chromatography column was started, such that the washing buffer in the third container 7 was allowed to flow through the third two-way solenoid valve 71 and then flow through the chromatography column 0 with the affinity medium, and a liquid flowing out after the washing-off was collected in the sixth container 10 through the silicone hose 2. The liquid level detector was always immersed in a liquid and was at a high electrical level throughout the washing process. About 66 min later, the liquid level detector was switched to a low electrical level, and 30 s later, the system completed the other protein removal operation of the chromatography column.

[0045] The system closed the third two-way solenoid valve 71 and opened the fourth two-way solenoid valve 81, and the silicone hose 2 was driven by the stepper motor 4 such that the silicone hose 2 was located directly above the fifth container 9 to collect a target protein. The continuous elution of the target protein for about 30 min was started, such that the elution buffer in the fourth container 8 was allowed to flow through the fourth two-way solenoid valve 81 and then flow through the chromatography column 0 with the affinity medium, and a liquid flowing out after the elution was collected in the fifth container 9 through the silicone hose 2 to obtain an eluted protein sample solution. Throughout the elution process, 30 s after the liquid level detector was switched from a high electrical level to a low electrical level once again, the system closed the peristaltic pump and all solenoid valves.

[0046] Through the above steps, the system fully automated the adsorption, washing, and elution of the target protein.