JPS6138457A - Method and apparatus for separating and purifying charged substance - Google Patents

Abstract

PURPOSE:To attain to enhance separation capacity, by recirculating a separation buffer solution in a separation chamber provided with a distribution plate and applying voltage toward the direction at a right angle to a recirculation stream while separating and purifying a charged substance from a charged substance solution by electrophoresis. CONSTITUTION:A cathode chamber 9 having a cathode 3 and an anode chamber 10 having an anode 4 are arranged to both sides of a separation chamber 1 in parallel and a distribution plate 18 having a large number of openings 20 provided to the central part thereof is arranged in parallel to the electrodes 3, 4 and an injection port 6 for supplying a protein solution 5 is provided to the upper of the central part. subsequently when the chamber 1 and both electrode chambers 9, 10 are filled with a separation buffer solution 2 and the temps. of the electrode chambers are set by temp. controllers. The buffer solution 2 is recirculated by a recirculation pump and the protein solution 5 is injected while voltage is applied between the electrodes 3, 4, protein is separated to either one of upper and lower takeout ports 7, 8 corresponding to the charge thereof.

Description

[Detailed Description of the Invention] [Field of Application of the Invention] The present invention relates to a technology for separating and purifying charged substances such as proteins, nucleic acids, and cells by electrophoresis, and in particular has high separation performance and can be used in large quantities. The present invention relates to a FIB purification method for charged substances that can be processed and an apparatus therefor.

[T of invention! Scenery]

Conventional methods for purifying charged substances such as rice grains, nucleic acids, and cells (hereinafter referred to as rice grains) include electrophoresis, membrane separation, liquid chromatography, and the like. Membrane separation is a method that separates rice grains by the size of the membrane's pores, and allows for continuous processing, but it has the disadvantage of poor separation ability for grains, while liquid chromatography is This method involves separation through a carrier-packed column, and although it has low separation performance, it is a batch operation and is therefore unsuitable for large-scale processing on an industrial scale.Also, electrophoresis is a method that separates proteins by passing them through a column packed with carriers. This electrophoresis method uses carriers such as gel to perform separation and purification in an electric field.This electrophoresis method includes carrier electrophoresis method that uses a carrier such as gel, and carrier-free electrophoresis method that is performed in a free-flowing liquid without using a carrier. The carrier electrophoresis method is the Pauch method, and the carrier-free electrophoresis method is suitable for industrialization where large-scale processing is performed.

For carrier-free electrophoresis, Electrophor
Kur in esis1982.3, 235-243
“New aspects” by T Hannig
in preliminary and analytical
ical continuous free-flow
In addition, an apparatus based on this principle has already been manufactured by Hirshmann in West Germany.This method for separating and purifying proteins using carrier-free electrophoresis is explained below. A mixture of proteins to be separated is continuously injected from an injection port into a separation buffer solution flowing at a constant speed across an electric field in a separation chamber. Each protein has a different amount of charge. As a result, their moving speeds in the electric field are different.Therefore, while flowing down the separation Puffer solution, they are deflected and separated depending on the flow rate of the separation buffer solution.

As described above, this method allows continuous separation and is therefore effective for industrial-scale separation and purification of rice grains.

In order to improve the separation performance with this method, it is important to keep the flow rate of the separation buffer solution in the separation chamber constant at all times, but since a current is passed through the separation buffer solution, Joule heat is always generated. This heat causes a convection phenomenon in the separation buffer solution, which disrupts the flow of the separation buffer solution, resulting in a decrease in the separation performance of rice grains. In order to solve this problem, in the conventional carrier-free electrophoresis method, the temperature and flow rate of the separated Päffer's solution are controlled with extremely high precision of ±0.2%, and the equipment is downsized and made compatible with Joule heat. However, the separation performance of rice grains is not very good.

Furthermore, in order to eliminate the effects of this thermal convection, there are plans to take an electrophoresis device into space and perform electrophoresis in zero gravity, which shows how big a problem this convection is.

[Purpose of the invention]

An object of the present invention is to provide a method for separating and purifying a charged substance, which has high separation performance and is capable of mass processing, and an apparatus therefor.

[Summary of the invention]

In conventional carrier-free electrophoresis devices, in order to improve separation performance, an important technical issue is how to control the convection of the separation buffer solution due to the generation of Joule heat and maintain a constant flow rate. For this reason, efforts have been made to strictly control the temperature and flow rate of the separation buffer solution, and to downsize the device.

The present inventors have conducted repeated experiments and research, focusing on the reverse use of the convection of separated Päffer's liquid due to the generation of Joule heat, which is unavoidable in electrophoresis, and as a result, they have arrived at the present invention. be.

The present invention is for separating and purifying a charged substance by treating a liquid containing a charged substance by electrophoresis, in which a circulating flow of a separation buffer solution is formed in a separation chamber in a direction perpendicular to the circulating flow of nucleic acids. The charged substance is separated and purified by applying a voltage to the separation chamber and supplying a charged substance solution into the separation chamber.

[Embodiments of the invention]

FIG. 1 shows the principle of the present invention, in which a circular flow is generated in a fixed direction in the buffer solution 2 in the separation chamber 1, and the negative type Mi! 3. A voltage is applied to the positive electrode 4 to form an electric field. Next, a liquid 5 containing the charged substance to be separated (hereinafter referred to as phlegm liquid) is supplied into the bone chamber 1 through the injection port 6. Here, it is assumed that the protein substance in the protein absorption liquid 5 is composed of protein substance (TIA, B), and protein substance B is negatively charged, and protein substance B is positively charged. The circulating flow of the separated Päffer's solution 2 is assumed to be in the direction of the arrow shown in the figure. The behavior of the roasting solutions A and H is that protein A is attracted to the positive electrode 4, and at the same time, due to the flow of the separation buffer solution 2, it is accumulated near the upper extraction lower. While being attracted to the negative electrode 3, the substance B is accumulated near the lower outlet 8 due to the flow of the separation buffer solution 2.

In this way, by utilizing the circulating flow of the separation buffer solution 2, it is possible to accurately separate and purify the rice grains (quality).

Hereinafter, one embodiment of the present invention will be described in detail with reference to FIG. In FIG. 2, l is a vertically installed separation chamber, 2 is a separation buffer solution, and 3 and q4 are a negative electrode chamber 9 and a positive electrode chamber 10 formed on both sides of the separation chamber 1.
A negative electrode and a positive electrode are provided in parallel within the separation chamber 1, 6 is an injection port for supplying the TI liquid 5 provided in the center of the separation chamber 1, and 7 is an anode chamber of the separation chamber 1. 8 is the upper outlet provided in the upper part of the IO side, 8 is the lower outlet provided in the cathode chamber 9 of the separation chamber 1, and 2 is the connection between the separation chamber 1 and the cathode chamber 9. Separated from the anode chamber 10
19 are temperature regulators for regulating the temperature in the negative J.r! chamber 9 and the positive electrode chamber 10, and 13 are temperature regulators for each temperature regulator. The separation buffer solution whose temperature was adjusted in step 12 is transferred to the cathode chamber 9.
and a circulation pump that circulates to the anode chamber 10.

Separation chamber 1 and cathode chamber 9. After filling the positive electrode chamber 10 with 1'+lt buffer solution, the negative electrode chamber 9
Internal temperature T, and positive? [The humidity temperature in the polar chamber IO is T, <
T.

Set each temperature adjustment needle-hill as desired.
When the separation buffer solution whose temperature has been adjusted by the circulation pump 13 is circulated to the Jitun Imitation Treasure 9 and the cathode chamber 1G, the separation buffer solution 2 in the separation chamber 1 is divided into the cathode chamber 9 and the anode electrode chamber 10. The temperature difference causes a circulating flow in the direction of the arrow in the figure. Next, while applying a voltage between the negative electrode 3 and the positive electrode 11117!4, the protein absorbent liquid 5 is injected into the separation chamber l from the injection port 6. 3.1? ) is attracted to the positive electrode 4 and separated into the upper extraction lower side and the lower extraction port 8 side.

Examples also used myoglobin (horse) as a protein sample with an isoelectric point of 7.1 to 7.3 and egg white lysozyme with an isoelectric point of 11.0 to 11.4.
While circulating the separation buffer solution in the separation chamber 1 with pH 9.0, a voltage was applied between the negative electrode 3 and the positive electrode 4 to form a solution in which the above two types of proteins were dissolved (PH=
9.0) was continuously supplied into the separation chamber 1 from the injection port 6 to perform electrophoresis. As a result, it was confirmed that the myoglobin solution was recovered from the upper extraction lower and the egg white lysozyme solution was recovered from the lower extraction port 8, and that the separation and purification of rice grains was carried out.

Figure v53 shows another embodiment of the present invention, and the same parts as in Figure 2 are designated by the same reference numerals, and their explanation will be omitted. In FIG. 3, 16 and 17 are the same as the electrophoresis device shown in FIG. The extraction port 8 and the injection port 6 of the second stage electrophoresis device 17 are PH:J.
! I2 tank 14. The protein solution is connected via a supply pump 15 and taken out from the lower outlet 8 of the first stage electrophoresis device a 16.

After the pH is adjusted in the pH adjustment tank 14, the supply pump 15
This allows the liquid to be supplied to the injection port 6 of the second stage electrophoresis device 17.

Examples and protein samples include albumin (bovine serum), isoelectric point 4.9 myoglobin (horse), isoelectric point 7.1-7.3 egg white lysozyme
Using an isoelectric point of 11.0 to 11.4, the pH value of one separation buffer solution is 6.0 for the first-stage electrophoresis apparatus 16 and 9.0 for the second-stage electrophoresis apparatus 17, respectively. While circulating the inside, the electricity protection [i3 and Yoden [! A voltage was applied between 4 and 4, and a solution (PH 6, 0) in which the three types of rice grains were dissolved was continuously supplied into the chamber 1 from the injection port 6 of the first stage electrophoresis device 7.16. Electrophoresis was performed. As a result, an albumin solution was recovered from the upper extraction lower, and a heated solution of a myoglobin solution and an egg white lysozyme solution was recovered from the lower extraction port 8.

After adjusting the pH of this mixed solution to 9.0 in the pH adjustment tank 14, it is transferred to the second stage electrophoresis device 17 by the supply pump 15.
The solution was continuously supplied into the separation chamber 1 from the injection port 6 of the cell, and electrophoresis was performed again. As a result, the myoglobin solution was recovered from the upper extraction lower, and the egg white lysodeme solution was recovered from the lower extraction port 8.

Example 3 Using the electrophoresis device shown in Figure 2, a diluted cytochrome C (bovine) solution with 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. While circulating the inside of the chamber l, the electrically-proof Mr! 3 and Yoden W
34, and the diluted cytochrome C solution (PH
= 8.0) was intermittently supplied into the separation chamber 1 from the injection port 6 to perform electrophoresis. As a result, a highly concentrated cytochrome C solution was recovered from the lower outlet port 8.

In the above embodiment, the separation chamber is reinstalled vertically, and a temperature difference is created between the negative electrode chamber 9 and the anode electrode chamber 10 to form a circulating flow of molecules and fur liquid in the separation chamber 1. However, even if the separation chamber is installed vertically or horizontally and electrophoresis is performed by mechanically forming a circulating flow of the separating buffer solution using a pump, a stirrer, etc., the same result as described above can be achieved. It is possible to separate and purify proteins with high performance.

〔Effect of the invention〕

As described above, the present invention forms a circulating flow of buffer solution in a separation chamber, applies a voltage in a direction perpendicular to the circulating flow of concentrated solution, and supplies a charged substance solution to the separation chamber. Since the charged substance lτ is supplied and separated and purified by electrophoresis, it is possible to ignore the convection of the separated Puffer solution due to Joule heat generated by passing an electric current through the separation buffer solution, and Since the flow rate of the separation buffer solution can always be kept constant, the amount of charged substances can be reduced by 1! In addition to improving the lIt performance, it also becomes possible to process charged substances with a large flt, which has great economical effects.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the principle of the present invention using carrier-free electrophoresis, and FIG. 2 is an electrophoresis IIl! diagram showing an embodiment of the present invention.
I! FIG. 3 is a schematic diagram of a two-stage electrophoresis device showing another embodiment of the present invention. 1... Separation chamber, 2... Minute 1'
llI buffer solution, 3... cathode, 4...
・・・Positive electrode, 5...Tanba ('ff (charged substance) liquid, 6...Bending inlet, 7...Bending/upper outlet, 8...
...Lower outlet, 9...Curved negative electrode chamber, 10:...
...Positive electrode chamber, 11... Separation membrane, 12... Four temperature regulators. 13... Circulation pump, 14... PHW
3! i tank, 15... bend supply pump, 16... 1st stage electrophoresis device 7l, 17...... Figure I bottom 7 V, central, exit Figure 12, procedure amendment ( (Spontaneous) Indication of the case Patent Application No. 158724 of 1982 Name of the invention Method for separation and purification of charged substances and its equipment ■ Person making the amendment and bookkeeper Name of the patent applicant Name Rshot4ShikikaiToF Kuchi Tate
Manufacturer, agent, person's amendment subject matter scope of claim column, detailed description of invention fence,
Contents of the amendment The column for the brief description of the drawings, Drawing 1, and the claims are amended as shown in the attached sheet. 2. Between the 9th and 10th lines from the top of page 13 of the specification, it says: ``Figure 4 shows yet another embodiment of the present invention, and the same parts as in Figure 2 are designated by the same reference numerals, and explanations will be given below. The description is omitted.
．． It is a rectifying plate placed parallel to Yoden 4Ir44, and has many openings on its entire surface so as not to obstruct electrophoresis, and cutouts at both ends so as not to obstruct the circulating flow of separated Babufa liquid 2. 19 are provided. Therefore, the current plate 18 may be made of a porous material such as a filter membrane, and may have cutouts 19 at both ends thereof. By providing the rectifying plate 18, it is possible to generate a stable circulation flow in the separation buffer 1 liquid 2 as indicated by the dotted line arrow in the figure, and the separation performance can be improved. ”
Add. 3. Details q! ) In the seventh line from the top of page 14, "Schematic diagram." is corrected to "Schematic diagram. FIG. 4 is a schematic diagram of electrophoresis*Fa+ showing still another embodiment of the present invention." 4. From the 14th purchase to the 15th line onwards, "18.
...Rectifier plate, 19...Notch, addition...
...opening," is added. 5. Claim 1, which is characterized by drawings and attached FIG. While circulating the puffer liquid,
A method for separating and purifying a charged substance, which comprises applying a voltage in a direction perpendicular to the circulating flow of nucleic acids and supplying a charged substance solution into a separation chamber to separate and purify the charged substance. 2. A method for separating and purifying a charged substance according to claim 1A1, wherein a circulating flow is formed by creating a temperature difference in the separation liquid in the separation chamber. 3. In a charged substance separation and purification device that processes a liquid containing a charged substance by electrophoresis to separate and purify the charged substance,
A negative electrode and a positive electrode are provided on both sides of the separation chamber, an injection port for a charged substance solution is provided in the separation chamber, and a charged substance solution is provided at one end of the negative electrode and the other end of the positive electrode of the separation chamber. An apparatus for separating and purifying a charged substance, characterized in that it is provided with an outlet and means for circulating the separated Babufur liquid in the separation chamber. 4. The separation chamber is vertically installed 71, and a negative electrode chamber and a positive electrode chamber are provided on both sides of the separation chamber with isolation membranes interposed therebetween as a means for circulating the separation Babfur liquid, and the negative electrode chamber and the positive electrode chamber i' [each in room ii
T! 1111j tZl-made apparatus for charged substances according to claim 3, which is provided with a temperature regulator and a circulation pump for circulating the If buffer solution. 5. In a charged substance separation device manufactured by t+'l, which processes a liquid containing a charged substance by electrophoresis to separate and purify the charged substance, there is a negative electrode and a positive electrode on both sides of the separation chamber. an inlet for a charged substance solution is provided in the separation chamber; an outlet for the charged substance is provided in one end of the negative electrode and the other end of the anode of the separation chamber; A rectifying plate with cutouts at both ends is arranged parallel to the negative electrode and the positive electrode in the center of the inside, and An apparatus for separating and purifying a charged substance, characterized by comprising a means for circulating a separated Babufur liquid in a 11F chamber. . 6. In a charged substance purification apparatus for separating and purifying charged substances by treating a liquid containing a charged substance by electrophoresis, a negative electrode and a positive electrode are provided on both sides of the separation chamber, and a chamber for the valve 111 is provided. An inlet for a charged substance solution is provided in the center of the separation chamber, and an outlet for the charged substance is provided at one end of the negative electrode and the other end of the anode of the separation chamber, and the separated Bavfer solution in the separation chamber is Multi-stage electrophoresis 4fi is performed by providing a plurality of electrophoresis apparatuses equipped with circulation means, and sequentially connecting the charged substance outlet of each electrophoresis apparatus to the charged substance solution injection port of the other electrophoresis apparatus.
A charged substance separation and purification device characterized by comprising a 'q. Age 4 [21]

Claims (1)

[Claims] 1. In a charged substance separation and purification method in which a liquid containing a charged substance is treated by electrophoresis to separate and purify the charged substance, while circulating a separation buffer solution in a separation chamber,
A method for separating and purifying a charged substance, which comprises applying a voltage in a direction perpendicular to the circulating flow of the liquid and supplying a charged substance solution into a separation chamber to separate and purify the charged substance. 2. The method for separating and purifying a charged substance according to claim 1, wherein a temperature difference is given to the separation buffer solution in the separation chamber to form a circulating flow. 3. In a charged substance separation and purification device that processes a liquid containing a charged substance by electrophoresis to separate and purify the charged substance,
A negative electrode and a positive electrode are provided on both sides of the separation chamber, an inlet for a charged substance solution is provided in the center of the separation chamber, and a charged substance solution is provided at one end of the negative electrode and the other end of the positive electrode of the separation chamber. An apparatus for separating and purifying a charged substance, characterized in that it is provided with a substance extraction port and means for circulating a separation buffer solution in the separation chamber. 4. The separation chamber is installed vertically, and as a means for circulating the separation buffer, a negative electrode chamber and a positive electrode chamber are provided on both sides of the separation chamber with a separation membrane interposed therebetween. 4. The apparatus for separating and purifying charged substances according to claim 3, further comprising a temperature regulator and a circulation pump for circulating a separation buffer solution in each electrode chamber. 5. In a charged substance separation and purification device that processes a liquid containing a charged substance by electrophoresis to separate and purify the charged substance,
A negative electrode and a positive electrode are provided on both sides of the separation chamber, an inlet for a charged substance solution is provided in the center of the separation chamber, and a charged substance solution is provided at one end of the negative electrode and the other end of the positive electrode of the separation chamber. A plurality of electrophoresis devices each having a substance outlet and a means for circulating the separation buffer solution in the separation chamber are provided, and the charged substance outlet of each electrophoresis device is sequentially connected to the other electrophoresis device. 1. A charged substance separation and purification device, characterized in that it is connected to an injection port for a charged substance solution to constitute a multistage electrophoresis device.