JPS6259853A - Separator for electric charge material - Google Patents

Abstract

PURPOSE:To continuously separate electric charge materials having specific isoelectric points and mol.wt. by disposing fractionation membranes having different pore sizes to the inside of a migration chamber in the direction where a DC voltage is impressed and providing outlet nozzles to chamber units at the bottom end of the liquid flow of the migration chamber. CONSTITUTION:The migration chamber 2 of a flow device body 1 is segmented to three chambers 2a, 2b, 2c by the fractionation membranes 5a, 5b which make fractionation according to the mol.wt. Electrodes 6a, 6b are respectively installed in electrode chambers 3a, 3b partitioned by a semipermeable membrane 4. The inlet and outlet nozzles 7a, 7b, 7c, 8a, 8b, 8c are respectively provided to the top and bottom ends of the migration chambers 2a, 2b, 2c. The mol.wt. to be held by the respective fractionation membranes is set at a prescribed value and the pH of a buffer soln. is adjusted to a prescribed value in the case of separating the specific protein from a mixture composed of proteins having respectively specific isoelectric points and mol.wt., then the specific protein moves to the specific electrode side with the relation between the isoelectric points and pH and is taken out of any of the nozzles 8a-8c.

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 protein absorbent, nucleic acids, cells, etc., and in particular, separation of charged substances using electrophoresis. This invention relates to a device for separating charged substances.

[Background of the invention]

Conventional methods for separating and purifying charged substances such as grains, nucleic acids, and cells include electrophoresis, membrane separation, and liquid chromatography. Membrane separation is a method that separates rice grains by the size of the membrane's pores, and allows for continuous treatment;
Liquid chromatography has the disadvantage of poor quality separation ability, and liquid chromatography is a method of separating protein absorbent through a column packed with a carrier, and although the separation ability is low, it is a batch operation that is unsuitable for industrial-scale mass processing. be. Furthermore, electrophoresis is a method of separating and purifying proteins in an electric field by utilizing differences in the amount of charge of proteins. This electrophoresis method includes a carrier electrophoresis method that uses a carrier such as a gel, and a carrier-free electrophoresis method that is carried out 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 where large-scale processing is performed.

Regarding the carrier-free electrophoresis method, Mr. Kurt Hanning of West Germany has published the document "Electrophoresis, 3. P235-243, 1982J (Ele
ctrophoresis, 3. P235-243
．． 1982) I have published this. The method is shown below.

The mixture to be separated is continuously injected from the injection port into the separation buffer flowing at a constant speed across the electric field in the electrophoresis chamber. The speeds are different.Therefore, while flowing through the separation buffer, they are deflected and separated depending on the flow rate of the separation buffer.In this way, this method can perform continuous separation, so it can be used in industrial-scale rice fields ( It can be seen that this method is effective for the separation and purification of quality.

In order to improve the separation performance in this method, it is important to always keep the flow rate of the separation buffer in the separation chamber constant. However, Joule heat is inevitably generated due to the current flowing through the separation buffer, and this heat causes a convection phenomenon in the separation buffer, disrupting the flow of the separation buffer. Therefore, the ability to separate proteins decreases. To improve this point, conventional carrier-free electrophoresis devices require extremely precise control of the temperature and flow rate of the separation buffer within ±0.2 inches. Unfortunately, the throughput is small, which is a problem when scaling up to an industrial scale.

[Purpose of the invention]

An object of the present invention is to provide a charged substance separation device that can perform isoelectric point fractionation and molecular weight fractionation in one device, and can continuously separate charged substances having a specific isoelectric point and molecular weight. It is to provide.

[JIA key points for invention]

The present invention applies a DC voltage across the electrophoresis chamber, causes a liquid to be processed containing a plurality of charged substances to flow into the electrophoresis chamber, and separates the charged substances by electrophoresis. A plurality of fractionation membranes each having a different pore size are arranged in the order of the pore size for the application method, and an outlet nozzle is provided for each chamber partitioned by the semipermeable membrane at the lower end of the liquid flow of the migration chamber. do.

[Embodiments of the invention]

An embodiment of the present invention will be described below with reference to Figures 1 and 2.

FIG. 1 is a front sectional view of a continuous separation device for charged substances according to the present invention, and FIG. 2 is a sectional view taken along the line 1-11 in FIG. In the figure, l is the flow, the movement device main body, 2 is the migration chamber, aa, 3
b is an electrode chamber. The migration chamber 2 and the electrode chambers 3a, 3b are separated by a semipermeable membrane 4, which is a fractionation membrane with a small pore size. The inside of the electrophoresis chamber 2 is divided into three chambers 2a, 2b, and 2c by fractions g5a and 5b, which are fractionated according to molecular weight. On the other hand, insides of the electrode chambers 3a and 3b are installed 6a and 6b for applying a DC voltage, respectively. At the upper end of the migration chamber 2C, there is an inlet nozzle 7C for a liquid to be processed containing a charged substance.
Separation liquid inlet nozzles 7a are provided at the upper ends of the migration chambers 2a and 2b,
There is 7b. At the lower ends of the migration chambers 2a, 2b, 2c are outlet nozzles 8a, 8b, 8c for the liquid to be processed. In addition, in the lower and upper parts of each of the electrode chambers 3a and 3b,
Electrode liquid inlet nozzles 9a, 9b and outlet nozzle 10a
.

10b is provided.

Here, as the semipermeable membrane 4 that partitions the electrode chambers 3a, 3b and the migration chamber 2, a reverse osmosis membrane or the like having a pore size that does not allow the passage of charged substances such as proteins is used. In addition, a membrane that maintains the molecular weight of the target component in the charged substance to be treated is used as the molecular weight fractionation membrane 5a that divides the inside of the flow chamber 2, and a molecule 1 fractionation membrane 5a is used.
For b, a membrane is used that allows the molecular weight of the target component to pass through, but does not allow a substance with a molecular weight larger than the target component to pass through.

The electrode solution (buffer) is poured into the electrode chambers 3a and 3b of the electrophoresis apparatus 1, the separation buffer is introduced from the upper part of the electrophoresis chamber za and 2b, and the protein and other substances are introduced from the upper part of the electrophoresis chamber 2c. A buffer solution containing a charged substance to be treated is introduced into the electrode rod 6a,
Apply DC voltage to 6b. Here, the pH of the buffer solution is adjusted to a value higher than the isoelectric point of the target component, and the electrode 6a is
When charged with 6b as negative, the charged substance with an isocenter higher than the pH of the buffer migrates in the direction of the negative electrode 6b, as shown by the locus 11d in the figure, and is transferred to the negative electrode side of the migration chamber 2c ( (on the right side of the figure) and is taken out from the outlet nozzle 8c. On the other hand, charged substances with isoelectric points lower than the pH of the buffer solution containing the target component migrate electrophoretically in the direction of the positive electrode 6a, as shown by trajectories 11 a , 11 b , and 11 C; Since the film films 5a and 5b are installed, a charged substance with a molecular weight larger than the target component is 11 c
As shown by the trajectory, it descends while remaining in the migration chamber 2C blocked by the molecular weight separation membrane 5b, and passes through the exit nozzle 8.
It flows out from C.

Further, as shown by the trajectory 11b, the target component passes through the molecular weight separation membrane 5b, is blocked by the next separation membrane 5a, descends in the migration chamber 2b, and is taken out from the exit nozzle 8b. Then, the charged substance, which has an isoelectric point lower than the pH of the buffer solution and is smaller than the target component, passes through both the separation membranes sb and sa and migrates to the left side of the figure, as shown by the trajectory 11a. The liquid is passed through the chamber 2aj and taken out from the outlet nozzle 8a.

In this way, the target component can be continuously extracted from a charged substance having a distribution in molecular weight and isoelectric point.

A case will be explained in which a mixture of commercially available proteins is separated using this apparatus. Myokinase (My.

kinase-=-・isoelectric point 4.31 minutes molecule 21,00
0) is a person.

Ova Ibum in...Isoelectric point 469 molecules B4s, ooo)
min-・-・-isoelectric point 4.7. Molecule fi57,000
) is C, triocephosphate isomerase (Tri
osephosphate isomerase 8゛-...isoelectric point 581 molecular weight 43,000) as D,
When trying to continuously separate B from these mixed solutions,
The molecular weight fractionation membrane 5a retains molecules ff140. Using an OOO membrane, the retention membrane 5b has a retention molecular weight of 60. Adjust the pH of the buffer to 52 using an OOO membrane. In this way, when a mixed solution containing Tanba<'FTA, B, C, and D is continuously supplied to this device I, a tank with an isofocus higher than the pH of the buffer solution (the mass is on the negative electrode side) is produced. In order to migrate, it descends through the migration chamber 2C along the trajectory 11d.Also, because the isoelectric point of the cell C is lower than the pH of the buffer solution, it migrates toward the positive electrode, but it does not reach the fractionation membrane 5b. The protein B is obstructed, moves down the migration chamber 2C along the trajectory 11c in the figure, and is taken out from the exit nozzle 8C together with the protein.The protein B has a positive polarity due to the relationship between the isoelectric point and the pH. It moves to the one-pole side, passes through the fractionation membrane 5b, is intercepted by the fraction @5a, moves down the migration chamber 2+) along the trajectory 11b, and is continuously taken out from the exit nozzle 8b.

On the other hand, when the rice field <WA moves to the positive electrode side, the fractionation membrane 5
b and 5a, as shown by locus 11a,
It descends within the migration chamber 2a and is taken out from the outlet nozzle 8a. In this way, proteins A, B, and C have equal distributions of points and molecular weights.

From the mixed solution of D, it is possible to continuously separate the target substance (quality B) of two molecules 14 with specific isoelectric points.

Another embodiment of the invention is shown in FIG. This example does not use the separation buffer inlet nozzle as shown in Fig. 1, and simplifies the device, and can exhibit sufficient performance in the case of a small device S7 with less processing 1. .

In the preferred embodiment of the present invention, as shown in FIG. 4, the pH of the buffer solution has a distribution in the charge direction, and the M buffer solution-filled nozzles 7a and 7a' are used.

This is a difference in P If of the liquid entering from 7b.

In this embodiment, the liquid to be treated that enters from the inlet nozzle 7C is first blocked by the molecular weight separation membrane 5b from passing through proteins larger than the target protein, and the proteins that have passed through the separation membrane 5b are charged in the charged direction. It migrates and descends to the equal point of each protein according to the pH distribution of
a, 8 a', 8b. At this time, the pore size of the fractions 11i5a and 5a' is adjusted by a knob according to the molecular weight of the target protein. As a result, proteins with a high molecular weight can be continuously separated, excluding proteins with large molecular weights.

Furthermore, in a preferred embodiment of the present invention, 41! of the separation buffer shown in factor @1! By changing the rate in the charging direction, the conductivity of the am liquid entering through the inlet nozzle 7a is increased, and the conductivity of the buffer solution entering through the inlet nozzle 7b is decreased. This is the fractionation membrane 5a from the jar.

The migration speed of protein in the migration chamber 2b partitioned by 5b can be increased, and the process 3! I! can be increased.

〔Effect of the invention〕

According to the present invention, charged substances having a specific isoelectric point and molecules
Continuous separation becomes possible.

[Brief explanation of drawings]

FIG. 1 is a longitudinal sectional view of an embodiment of the present invention, and FIG. 2 is a sectional view taken along line 11--H in FIG. FIGS. 3 and 4 each show other embodiments of the present invention. 1...Migration apparatus main body, 2...Migration chamber,
3a, 3b...electrode chamber, 4...semipermeable membrane, 5a, 5b...tetradome fractionation membrane

Claims (1)

[Claims] 1. A charged substance separation device that separates the charged substances by supplying a liquid to be processed containing a plurality of charged substances into a migration chamber to which a DC voltage is applied in a direction perpendicular to the flowing direction of the liquid to be processed, A plurality of fractionation membranes each having a different pore size are arranged in the direction of application of the DC voltage in the electrophoresis chamber in the order of the pore size, and an outlet nozzle is provided for each chamber partitioned by the semipermeable membrane at the lower end of the liquid flow of the electrophoresis chamber. A charged substance separation device characterized by being provided with: