JPH0715454B2 - Method and apparatus for separating and purifying charged substances - Google Patents

Description

TECHNICAL FIELD The present invention relates to a technique for separating and purifying charged substances such as proteins, nucleic acids, cells, etc. by an electrophoretic method, which has a particularly high separation performance and is suitable for large-scale processing. The present invention relates to a method for separating and purifying possible charged substances and an apparatus therefor.

[Background of the Invention]

Conventional methods for separating and purifying charged substances such as proteins, nucleic acids, and cells (hereinafter, typically referred to as proteins) include electrophoresis, membrane separation, and liquid chromatography. Membrane separation is a method of separating proteins according to the size of the pores of the membrane, and continuous treatment is possible, but it has the disadvantage of poor protein separation performance.Liquid chromatography is a method of separating proteins through a carrier-filled column. Although it has excellent separation ability, it is not suitable for industrial-scale large-scale processing because it is a batch operation. The electrophoresis method is a method of separating and purifying in an electric field by utilizing the difference in the charge amount of proteins. This electrophoresis method includes a carrier electrophoresis method using a carrier such as a gel and a carrier-free electrophoresis method performed in a free flowing liquid without using a carrier. The carrier electrophoresis method is a batch method, and the carrier-free electrophoresis method is suitable for industrialization in which a large amount of treatment is performed.

For carrier-free electrophoresis, see Electrophores-is 1982,
Kurt Hannig on “New aspccts in 3,235-243”
praparative and analytical continuous free-flow c
ell Clectrophoresis ", and a device based on this principle was found in Hirshman, West Germany.
Already manufactured by n companies. A method for separating and purifying a protein by the carrier-free electrophoresis method will be described. A mixture of proteins to be separated is continuously injected from an injection port into a separation buffer solution that flows at a constant rate across an electric field in a separation chamber. Since each protein has a different charge amount, the moving speed in an electric field is different. Therefore, while flowing in the separation buffer solution, it is deflected and separated depending on the flow rate of the separation buffer solution. As described above, the present method allows continuous separation, and thus is effective for industrial-scale protein separation and purification.

In order to improve the separation performance with this method, it is important to always keep the flow rate of the separation buffer solution in the separation chamber constant, but since an electric current is passed through the separation buffer solution, Joule heat is always generated, This heat causes a convection phenomenon in the separation buffer solution, and the flow of the separation buffer solution is disturbed, so that the protein separation performance deteriorates. In order to solve this problem, in the conventional carrier-free electrophoresis method, the temperature and flow rate of the separation buffer solution are controlled with extremely high accuracy of ± 0.2%, and the device is downsized to support Joule heat. , Protein separation performance is not so good.

Furthermore, there is a plan to bring the electrophoretic device to outer space to eliminate the influence of this thermal convection, and to perform electrophoresis in a weightless state, and you can see how serious this convection is.

[Object of the Invention]

An object of the present invention is to provide a method for separating and purifying a charged substance having high separation performance and capable of large-scale processing, and an apparatus therefor.

[Outline of Invention]

In the conventional carrier-free electrophoresis apparatus, in order to improve the separation performance, how to prevent convection of the separation buffer solution due to generation of Joule heat and maintain a constant flow rate is an important technical issue. Therefore, conventionally, the temperature and flow velocity of the separation buffer solution have been strictly controlled and the device has been downsized.

The present inventors have conducted experiments and research focusing on the converse use of convection of the separation buffer solution due to the generation of Joule heat, which is unavoidable in the electrophoresis method, and as a result, have reached the present invention. is there.

The present invention relates to a method in which a liquid containing a charged substance is treated by an electrophoretic method to separate and purify a charged substance. In the separation chamber, a circulating flow of a separation buffer liquid is formed, and the circulating flow of the liquid is perpendicular to the liquid. A voltage is applied in the direction, and a charged substance solution is supplied into the separation chamber to separate and purify the charged substance.

Example of Invention

FIG. 1 shows the principle of the present invention, in which a circulating flow in a certain direction is generated in a separation buffer solution 2 in a separation chamber 1, and a voltage is applied to a negative electrode 3 and a positive electrode 4 to form an electric field. . Next, a liquid 5 containing a charged substance to be separated (hereinafter referred to as a protein liquid) 5 is supplied into the separation chamber 1 through an injection port 6. Here, the protein in protein liquid 5 is
It is assumed that it is composed of proteins A and B, protein A is negatively charged, and protein B is positively charged. Further, the circulation flow of the separation buffer solution 2 is a flow in the direction of the arrow shown. Protein solutions A and B in the protein solution 5 supplied into the separation chamber 1
In the behavior of, the protein A is attracted to the positive electrode 4 and, at the same time, is accumulated in the vicinity of the upper outlet 7 in consideration of the flow of the separation buffer solution 2. Further, the protein B is accumulated in the vicinity of the lower outlet 8 while being attracted to the negative electrode 3 in consideration of the flow of the separation buffer solution 2.

In this way, the circulating flow of the separation buffer solution 2 can be utilized to accurately separate and purify proteins.

An embodiment of the present invention will be described in detail below with reference to FIG. In FIG. 2, 1 is a vertical separation chamber, 2 is a separation buffer solution, and 3 and 4 are negative electrode chambers 9 and positive electrode chambers 10 formed on both sides of the separation chamber 1.
A negative electrode and a positive electrode provided in parallel in the inside, 6 is an injection port provided in the central portion of the separation chamber 1 for supplying the protein liquid 5, and 7 is a positive electrode chamber 10 of the separation chamber 1.
An upper outlet provided on the upper side, 8 is a separation chamber 1
A lower outlet provided on the lower side of the negative electrode chamber 9 side, 11 is an isolation membrane made of a member that separates the separation chamber 1 from the negative electrode chamber 9 and the positive electrode chamber 10 and passes a current, and 12 is the negative electrode chamber, respectively. 9
And a temperature controller for adjusting the temperature in the positive electrode chamber 10, and 13 is a circulation pump for circulating the separation buffer solution whose temperature is adjusted by each temperature controller 12 to the negative electrode chamber 9 and the positive electrode chamber 10, respectively. .

Separation chamber 1 and in the cathode chamber 9, after filling the separation buffer solution into the anode chamber 10, cathode chamber 9 in the temperature T ₉ and anode chamber 10 inside the temperature T ₁₀ is T ₉ <T ₁₀ As described above, the respective temperature adjusters 12 are set so that the separation buffer solution whose temperature is adjusted by the circulation pump 13 is supplied to the negative electrode chamber 9 and the negative electrode chamber 10.
When it is circulated, the separation buffer solution 2 in the separation chamber 1 produces a circulating flow in the direction of the arrow in the figure due to the temperature difference between the negative electrode chamber 9 and the positive electrode chamber 10. Next, the negative electrode 3 and the positive electrode 4
By injecting the protein solution 5 into the separation chamber 1 through the inlet 6 while applying a voltage between them, the protein solution 5 flows together with the flow of the separation buffer solution 2, and the protein is charged by the negative electrode 3 depending on its charge. Alternatively, it is sucked by the positive electrode 4 and separated into the upper outlet 7 side and the lower outlet 8 side.

Example 1 Using a myoglobin (horse) isoelectric point of 7.1 to 7.3 egg white lysozyme and an isoelectric point of 11.0 to 11.4 as a protein sample, the pH of the separation buffer solution was set to 9.0, and the negative electrode 3 and positive electrode were circulated in the separation chamber 1. A liquid in which the above two types of proteins are dissolved by applying a voltage between the electrodes 4 (PH =
9.0) was continuously supplied from the inlet 6 into the separation chamber 1 for electrophoresis. As a result, the upper outlet 7
It was confirmed that the myoglobin solution was recovered, the egg white lysozyme solution was recovered from the lower outlet 8, and the protein was separated and purified.

FIG. 3 shows another embodiment of the present invention. The same parts as those in FIG. 2 are designated by the same reference numerals, and the description thereof will be omitted. In FIG. 3, 16 and 17 are a first-stage electrophoresis device and a second-stage electrophoresis device having the same structure as the electrophoresis device shown in FIG. The injection port 6 of the second-stage electrophoresis device 17 is the PH adjusting tank 14 and the supply pump 15.
The protein solution extracted from the lower outlet 8 of the first-stage electrophoresis device 16 is connected to the pH adjusting tank.
The pH is adjusted in 14 and then supplied to the inlet 6 of the second-stage electrophoresis device 17 by the supply pump 15.

Example 2 An albumin (bovine serum) isoelectric point 4.9 myoglobin (horse) isoelectric point 7.1 to 7.3 egg white lysozyme isoelectric point 11.0 to 11.4 was used as a protein sample, and the PH value of the separation buffer solution was the first stage electrophoresis apparatus 16 6.0 For the second-stage electrophoresis apparatus 17, 9.0 is a solution in which the above three kinds of proteins are dissolved by applying a voltage between the negative electrode 3 and the positive electrode 4 while circulating in the separation chamber 1 (PH6.0). ) Was continuously supplied from the inlet 6 of the first-stage electrophoresis device 16 into the separation chamber 1 for electrophoresis. As a result, the albumin solution was recovered from the upper outlet 7, and the mixed solution of the myoglobin solution and egg white lysozyme solution was recovered from the lower outlet 8. The pH of the mixed solution is adjusted to 9.0 by the PH adjusting tank 14, and then the supply pump 15 continuously supplies the mixed solution into the separation chamber 1 through the inlet 6 of the second-stage electrophoresis apparatus 17 to perform electrophoresis again. Let As a result, the myoglobin solution was recovered from the upper outlet 7, and the egg white lysozyme solution was recovered from the lower outlet 8.

Example 3 Used in the electrophoresis apparatus shown in FIG. 2, a diluted cytochrome C (bovine) solution having an isoelectric point of 10.6 was used as a protein sample, and the pH of the separation buffer solution was set to 8.0 and circulated in the separation chamber 1. Meanwhile, a voltage was applied between the negative electrode 3 and the positive electrode 4, and the diluted cytochrome C solution (PH = 8.0) was intermittently supplied from the injection port 6 into the separation chamber 1 for electrophoresis. As a result, a high concentration cytochrome C solution was recovered from the lower outlet 8.

In the above-described embodiment, the separation chamber is installed vertically, and the temperature difference is provided between the negative electrode chamber 9 and the positive electrode chamber 10 to form the circulation flow of the separation buffer solution in the separation chamber 1. However, even if the separation chamber is installed vertically or horizontally, and a circulation flow of the separation buffer solution is mechanically formed using a pump, a stirrer or the like to perform electrophoresis,
Similar to the above, the protein can be separated and purified with high performance.

〔The invention's effect〕

As described above, the present invention forms a circulation flow of the separation buffer solution in the separation chamber, applies a voltage in a direction perpendicular to the circulation flow of the separation solution, and supplies the charged substance solution into the separation chamber. Since the charged substance is separated and purified by electrophoresis, the convection of the separation buffer solution due to Joule heat generated by passing an electric current through the separation buffer solution can be ignored, and the separation buffer solution can be ignored. Since it is possible to always keep the flow rate constant, the performance of separating charged substances can be improved, and a large amount of charged substances can be processed, which is economically advantageous.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the principle of the present invention by a carrier-free electrophoresis method, FIG. 2 is a schematic diagram of an electrophoresis apparatus showing one embodiment of the present invention, and FIG. 3 is another embodiment of the present invention. 1 is a schematic diagram of a two-stage electrophoresis device. 1 ... Separation chamber, 2 ... Separation buffer solution, 3 ...
… Negative electrode, 4 …… Positive electrode, 5 …… Protein (charged substance) liquid, 6 …… Injection port, 7 …… Upper outlet, 8 …… Lower outlet, 9 …… Negative electrode chamber, 10 …… Positive electrode chamber, 11 …… Separator membrane, 12 …… Temperature controller, 13 …… Circulation pump, 14 …… PH adjusting tank, 15 …… Supply pump, 16 …… First stage electrophoresis device,
17 …… Second-stage electrophoresis device

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nakatsu Fujitsuji, 502 Jinritsucho, Tsuchiura-shi, Ibaraki Prefecture, Hiritsu Seisakusho Co., Ltd. (72) Inventor, Kiyoshi Fujiwara, Higashi-Toyoi, Daimo, Shimomatsu, Yamaguchi Prefecture, Ltd., Kasado Plant, Hitachi, Ltd.

Claims (5)

[Claims] 1. A method of separating and purifying a charged substance, which comprises treating a liquid containing a charged substance by an electrophoresis method to separate and purify the charged substance, while circulating a separation buffer solution in a separation chamber while circulating the liquid. Apply a voltage at a right angle to
A method for separating and purifying a charged substance, comprising supplying a charged substance solution into a separation chamber to separate and purify the charged substance. 2. The method according to claim 1, wherein the separation buffer solution in the separation chamber has a temperature difference to form a circulating flow.
A method for separating and purifying a charged substance according to the item. 3. An apparatus for separating and purifying a charged substance, wherein a liquid containing a charged substance is treated by electrophoresis to separate and purify the charged substance, wherein a negative electrode and a positive electrode are provided on both sides of the separation chamber, and the separation chamber is provided. An inlet for the charged substance solution is provided on one side, an outlet for the charged substance is provided on one end side of the negative electrode and the other end side of the positive electrode of the separation chamber, and means for circulating the separation buffer solution in the separation chamber is provided. An apparatus for separating and purifying a charged substance, comprising: 4. The negative electrode chamber and the positive electrode chamber are provided on both sides of the separation chamber via a separating film as means for circulating the separation buffer solution by vertically installing the separation chamber, The apparatus for separating and purifying a charged substance according to claim 3, wherein a temperature controller and a circulation pump for circulating the separation buffer solution are provided in the chamber and the positive electrode chamber, respectively. 5. An apparatus for separating and purifying a charged substance, wherein a liquid containing a charged substance is treated by electrophoresis to separate and purify the charged substance, wherein a negative electrode and a positive electrode are provided on both sides of the separation chamber, and the separation chamber is provided. An inlet for the charged substance solution is provided at the center of the separation chamber, an outlet for the charged substance is provided at one end side of the negative electrode and the other end side of the positive electrode of the separation chamber, and the separation buffer solution in the separation chamber is circulated. A plurality of electrophoretic devices provided with a means for allowing the electrophoretic devices to be provided, and the outlet for the charged substance of each of the electrophoretic devices is sequentially connected to the inlet for the charged substance solution of another electrophoretic device to form a multi-stage electrophoretic device An apparatus for separating and purifying charged substances, characterized in that.