JP3359370B2 - Electrophoresis gel for separation and recovery and separation and recovery method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gel used as an electrophoresis support. The gel of the present invention can be suitably used for separation and recovery of DNA (deoxyribonucleic acid), protein and the like.

[0002]

2. Description of the Related Art In recent years, proteins and nucleic acids (DNA or RNA (ribonucleic acid)) or fragments thereof have been analyzed for amino acid analysis, peptide mapping, amino acid sequencing, antibody production, gene cloning, DNA base sequence determination, and the like. BACKGROUND ART For production of recombinant DNAs, separation and recovery by electrophoresis are actively performed.

As a method of separation and recovery by electrophoresis, for example, a gel containing a substance to be separated separated by electrophoresis is cut out, and an electric field is applied again to elute the substance to be separated in the gel; A blotting method in which a filter paper or a nitrocellulose membrane is stacked on a gel containing a separation substance and the separation target substance is transferred to these carriers; a gel crushing method in which the gel is crushed to extract the separation target substance; A gel dissolving method for eluting a substance to be separated in a gel has been developed.

However, regarding these methods,
It has been pointed out that the recovery operation is complicated, the number of steps is large, the recovery rate is low, and the sample is easily denatured. In particular, when a small amount of protein or DNA is separated and recovered, a low recovery rate is fatal.

[0005] Among the above methods, the gel dissolving method has a feature that the recovery rate is relatively high, but it is necessary to use a gel material that can easily dissolve the gel.

[0006] Polyacrylamide gels or agarose gels are commonly used as supports for electrophoresis. Among these, polyacrylamide gel is
Since polyacrylamide is chemically crosslinked via N, N'-methylenebisacrylamide via a covalent bond, it cannot be normally dissolved. Therefore, instead of N, N'-methylenebisacrylamide as a crosslinking agent,
Degradable crosslinkers that are relatively easily decomposed, such as N,
N'-diallyltartaramide diamide, N, N '-(1,2-
Dihydroxyethylene) bis-acrylamide, N,
Soluble polyacrylamide gels using N'-bis-acrylcystamine and the like have been proposed (for example, J. T.
as et al., Analytical Biochemistry, 100: 264-270, 1979.
).

However, in dissolving the soluble polyacrylamide gel, it is necessary to decompose the cross-linking agent by a chemical reaction such as hydrolysis, oxidation, or reduction, and therefore, there is a problem of denaturation of the substance to be separated in the gel.

[0008] On the other hand, agarose gel exhibits a thermoreversible sol-gel transition that forms a gel at low temperatures and an aqueous solution at high temperatures.
Although it can be dissolved by heating, the melting point of ordinary agarose gel is extremely high, about 90 ° C. or higher. Therefore, low melting point agarose has been developed in which agarose is subjected to chemical modification such as hydroxyethylation to reduce the melting point (for example, L. Wieslander, Analytical Biochemistry, 98: 3).
05, 1979).

However, even with this low-melting-point agarose, the melting point is as high as about 60 ° C. or more, and it has been difficult to recover a sample that is easily denatured by heat. Therefore, this low-melting-point agarose is a sample that is hardly denatured even at a relatively high temperature.
It has been mainly used only for separation and recovery of A and RNA,
Since agarose is a naturally-derived material, it contains various impurities, and it has been pointed out that these impurities inhibit the activities of various enzymes (DNA ligase, restriction enzymes, etc.) used in the process of cloning DNA molecules. ing.

[0010] Another problem common to the gel dissolving method is that almost 100% of the substance to be separated can be recovered when the gel is dissolved, but the separated solubilized gel material and the substance to be separated are separated thereafter. In some cases, the recovery rate may be reduced during the process (eg, DC Filter and SS
Tevethia, Virology, 117: 267-270, 1982).

Therefore, it is very difficult to recover a substance to be separated from a gel by a simple method and at a high recovery rate in an undenatured state even if separation is performed by electrophoresis in the conventional method.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel electrophoresis gel for separation and recovery which can solve the above-mentioned problems of the conventional electrophoresis gel.

Another object of the present invention is to provide a separation / recovery method capable of obtaining a desired substance such as protein or DNA at a high recovery rate by a simple process using such an electrophoresis gel for separation / recovery. It is in.

[0014]

The object of the present invention is to provide a thermo-reversible sol-gel transition which forms a sol at a temperature lower than the sol-gel transition temperature and a gel at a temperature higher than the sol-gel transition temperature. Achieved by a distinctive electrophoretic gel for separation and recovery.

According to the present invention, the substance is separated by electrophoresis using the above-mentioned electrophoretic gel for separation and recovery in a gel state at a temperature higher than the sol-gel transition temperature, and then the gel is separated from the sol-gel. A separation / recovery method is provided in which a sol is formed by cooling to a temperature lower than the transition temperature, and the separated substance is recovered.

According to the present invention, further, the substance is separated by electrophoresis using an electrophoresis gel for ethyl alcohol-soluble separation and recovery in a gel state at a temperature higher than the sol-gel transition temperature as a support, and then the gel is subjected to ethyl separation. There is provided a separation and recovery method characterized by recovering the separated substance dissolved in alcohol.

Hereinafter, the present invention will be described in detail.

(Sol / Gel) The sol in the present invention is
An aqueous solution or a colloidal aqueous suspension of a polymer compound, which means that they have fluidity. On the other hand, a gel refers to a state in which the sol solidifies and loses fluidity. When the solvent used with the gel of the present invention is water, such a gel is generally called a hydrogel.

(Sol-gel transition temperature) In the present invention, the "sol state", "gel state" and "sol-gel transition temperature" are defined as follows. This definition is described in the literature (Polymer Journal. 18 (5), 411-416 (1)
986)).

1 mL of the polymer solution is placed in a test tube having an inner diameter of 1 cm, and kept in a water bath at a predetermined temperature (constant temperature) for 12 hours. Thereafter, when the test tube is turned upside down, if the solution / air interface (meniscus) is deformed by the weight of the solution (including the case where the solution flows out), the polymer solution is not allowed to flow at the predetermined temperature. Defined as "sol state".
On the other hand, the above solution /
When the air interface (meniscus) is not deformed by the weight of the solution, the solution is in a “gel state” at the above-mentioned predetermined temperature.
Is defined as

On the other hand, in the above measurement, the concentration was about 5 wt.
%, The temperature at which the “sol state” transitions to the “gel state” is determined by gradually increasing the above “predetermined temperature” (for example, at 1 ° C. increments). The temperature is defined as a “sol-gel transition temperature” (at this time, the “predetermined temperature” may be lowered in steps of, for example, 1 ° C., and the temperature at which the “gel state” transitions to the “sol state” may be obtained).

The gel used in the present invention is in a sol state at a temperature lower than the sol-gel transition temperature, is in a gel state at a temperature higher than the sol-gel transition temperature, and returns to the sol by cooling, that is, the sol -Has the property that the gel transition occurs thermoreversibly. The temperature at which this sol-gel transition occurs is the above-mentioned sol-gel transition temperature. In the present invention, the sol-gel transition temperature is 0 ° C.
The temperature is preferably from 90 ° C to 90 ° C, more preferably from 0 ° C to 40 ° C.

(Gel Material) Examples of the gel material having the above properties include cellulose ethers such as methylcellulose and hydroxypropylmethylcellulose (N. Sarkar, Journal of Applied Polymer Science, 2).
4: 1073-1087, 1979), chitosan derivatives (KR Holme
And LD Hall, Macromolecules, 24: 3828-3833, 199
1) Polysaccharide derivatives such as; block copolymers of polypropylene oxide and polyethylene oxide (SC Mill
er and MD Donovan, International Journal of Pharm
aceutics, 12: 147-152, 1982); polymethacrylic acid (J. Eliassaf and A. Silberberg,
Polymer, 3: 555-564, 1962).

Further, according to the knowledge of the present inventors, it is a block copolymer comprising a polymer compound having a cloud point and another water-soluble polymer compound, wherein the block copolymer has the cloud point in the block copolymer. A gel having the above properties can be obtained by using a material having a plurality of high molecular compounds. Here, the cloud point refers to a temperature at which clouding occurs for the first time when an aqueous solution (concentration: 1 wt%) of a polymer compound having a cloud point is gradually heated. This cloud point is described in the literature (J. Phys. Che
m., 93 , 3311 (1989)). In the present invention, the cloud point is 0 ° C. to 90 ° C., more preferably 0 ° C.
It is preferable that it is -40 degreeC.

(Polymer Compound Having Cloud Point) Examples of the “polymer compound having a cloud point” used in the present invention include poly N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives, and copolymers thereof. , Polypropylene oxide, polyvinyl methyl ether, partially acetylated polyvinyl alcohol, and the like.

(Water-soluble polymer) The above-mentioned "other water-soluble polymer" in the present invention includes, for example, dextran, polyethylene oxide, polyvinyl alcohol, poly-N-
Vinylpyrrolidone, polyvinylpyridine, polyacrylamide, polymethacrylamide, polyN-methylacrylamide, polyhydroxymethylacrylate, polyacrylic acid, polymethacrylic acid, polyvinylsulfonic acid, polystyrenesulfonic acid and salts thereof, polyN, N-dimethyl Examples include aminoethyl methacrylate, poly N, N-diethylaminoethyl methacrylate, poly N, N-dimethylaminopropylacrylamide, and salts thereof.

(Block Copolymer) The synthesis of the block copolymer consisting of the “polymer compound having a cloud point” and the “other water-soluble polymer compound” may be performed, for example, by using a “polymer compound having a cloud point”. And the monomer constituting "another water-soluble polymer compound" are repeatedly and sequentially polymerized.

More specifically, for example, when the polymer compound having a cloud point is polypropylene oxide and the other water-soluble polymer compound is polyethylene oxide, propylene oxide and ethylene oxide are anion-polymerized or cationic-polymerized. Are repeatedly and sequentially polymerized to obtain a block copolymer in which polypropylene oxide and polyethylene oxide are bonded.

The block copolymer of the present invention can also be obtained by connecting a material such as Pluronic (trade name, manufactured by BASF) F-127 in which polyethylene oxide is bonded to both ends of polypropylene oxide. it can.

The synthesis of a block copolymer consisting of a polymer having a cloud point and another water-soluble polymer is as follows:
It can also be obtained by introducing a polymerizable group (for example, an acryl group) into a polymer compound having a cloud point and then copolymerizing a monomer constituting the water-soluble polymer. It can also be obtained by introducing a polymerizable group therein and then copolymerizing a monomer constituting a polymer compound having a cloud point. Also, in the water-soluble polymer,
A functional group (eg, carboxyl group) capable of binding reaction with a functional group (eg, amino group) in a polymer compound having a cloud point
And reacting the two to react with each other.

The inventor of the present invention has found that the block copolymer of the present invention obtained by the above-mentioned method is water-soluble at a temperature lower than the cloud point of a polymer compound having a cloud point in the molecule, and is a homogeneous aqueous solution. However, it was found that at temperatures higher than the cloud point, the aqueous solution lost its fluidity and became a hydrogel. Furthermore, according to the findings of the present inventor, when a material in which a plurality of polymer compounds having the cloud point are present in the block copolymer is used, the hydrogel has a temperature higher than the cloud point. More water (e.g. 10-1 by weight)
The hydrogel did not dissolve even when water (00 times the amount of water) was added.

The content of the “polymer compound having a cloud point” in the block copolymer of the present invention is about 5 wt%.
Preferably, it is in the range of from about 90% by weight to about 90% by weight, more preferably from 30 to 70% by weight. If the abundance is less than 5% by weight, gelation becomes difficult, and if it is more than 90% by weight, the water content of the hydrogel becomes low, making it unsuitable as an electrophoresis support.

(Ethyl Alcohol Soluble) Polyacrylamide and agarose, which have been conventionally used as an electrophoresis support, are both insoluble in ethyl alcohol. However, in the electrophoresis gel for separation and recovery of the present invention, the gel is used. Ethyl alcohol-soluble material can be used as a constituent material.

For example, as described above, when the “polymer compound having a cloud point” is polypropylene oxide and the “other water-soluble polymer compound” is polyethylene oxide, both polymers are soluble in ethyl alcohol.
As a result, their block copolymers are also soluble in ethyl alcohol. In general, a sample separated by electrophoresis is a charged substance, and a sample having many charges has high water solubility but is insoluble in ethyl alcohol in many cases. Therefore, when collecting a sample, as represented by DNA,
A method of precipitating a sample with ethyl alcohol is widely used. Here, if the gel material is soluble in ethyl alcohol as described above, the sample to be separated and the gel material can be easily separated using the difference in solubility in ethyl alcohol.

In the present invention, when the material constituting the gel is soluble in ethyl alcohol, the gel material is
It is preferable to dissolve 10 g or more (more preferably, 50 g or more) in 100 ml of ethyl alcohol at ° C.

(Sample Separation / Recovery Method) Next, a preferred embodiment of a method for separating and recovering samples such as proteins and DNAs using the separation / recovery electrophoresis gel of the present invention will be described.

First, the gel material of the present invention is dissolved in a predetermined buffer for electrophoresis at a temperature lower than its sol-gel transition temperature. The aqueous solution is poured into a mold having a desired shape and gelled at a temperature higher than the sol-gel transition temperature of the gel material. As this mold, a normal electrophoresis apparatus can be used as it is, and a lane can be formed by inserting a comb before gelation. In the following operation, it is necessary to perform electrophoresis while maintaining the gel at a temperature higher than the sol-gel transition temperature of the gel, but otherwise perform electrophoretic separation of the sample in exactly the same operation as in normal gel electrophoresis. It is possible.

Thereafter, the gel portion containing the target sample separated by the electrophoresis method is cut out and cooled to a temperature lower than the sol-gel transition temperature of the gel, thereby dissolving (soling) the gel. Separation of the dissolved gel from the target sample
It can be carried out by a method usually used for collecting a sample from an agarose gel, for example, chromatography. (Ethyl Alcohol Precipitation Method) In the present invention, when the above gel material is soluble in ethyl alcohol, the gel is dissolved in ethyl alcohol, and the target sample is precipitated and precipitated with ethyl alcohol. The target sample can be easily separated.

In this ethyl alcohol precipitation method, for example, P
D amplified by the CR (polymerase chain reaction) method or the like
NA is purified using the separation and recovery electrophoresis gel of the present invention,
It is suitably used when collecting.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[0041]

Example 1 (Preparation of electrophoresis gel for separation and recovery) To 1 mol of trimethylolpropane, 160 mol of ethylene oxide was added by cationic polymerization to obtain polyethylene oxide triol having an average molecular weight of about 7,000. 100 g of this polyethylene oxide triol is distilled water 1000 m
Then, 12 g of potassium permanganate was gradually added at room temperature, and an oxidation reaction was carried out for about 1 hour. After filtration, the product was extracted into chloroform, and the solvent (chloroform) was distilled off under reduced pressure to obtain 90 g of a polyethylene oxide tricarboxyl compound.

10 g of the polyethylene oxide tricarboxyl compound and a polypropylene oxide diamino compound (average degree of polymerization of propylene oxide: about 65, manufactured by Jefferson Chemical Company, USA: trade name: Jeffamine D-40)
00, cloud point: about 9 ° C.) 10 g with carbon tetrachloride 1000 m
1 g of dicyclohexylcarbodiimide
Was added, and the mixture was reacted at the boiling point reflux for 6 hours. After cooling and filtering the reaction solution, the solvent was distilled off under reduced pressure, and the residue was dried under vacuum.
A block copolymer composed of polypropylene oxide and polyethylene oxide was obtained.

The block copolymer thus obtained was subjected to a 40 mM Tris-acetate buffer (pH 8.1, 1 mM EDTA and 0.5 μg / m 2) under ice-cooling at a concentration of 7 wt%.
l of ethidium bromide)
Was dissolved. This aqueous solution was poured into a UV transmission rack of a horizontal electrophoresis apparatus (manufactured by Marisol, model KS-8426) and gelled at room temperature with a comb (slot former).

Example 2 (Electrophoretic Separation and Recovery of DNA) The electrode tank was filled with the above-mentioned Tris-acetate buffer, and one of the sample grooves (lanes) from which the comb was removed was provided with a DNA molecular weight marker (restriction enzyme HaeIII of φX174 DNA) Digested material, Nippon Gene)
Was added to another lane in an amount of 0.5 μg of plasmid DNA (pUC19, 2.7 kilobase pairs, manufactured by Takara) linearized by digestion with the restriction enzyme EcoRI.
Run for hours.

After the electrophoresis, the separation pattern of the DNA marker obtained by fluorescence detection was the same as that of a normal agarose gel (concentration: 2%). On the other hand, the gel of the part considered to contain the plasmid DNA based on the fluorescence detection is about 0.
1 g was cut out, and the gel was dissolved by ice cooling, and 1 ml of ethyl alcohol was added to precipitate a precipitate. After recovering this precipitate by centrifugation (12,000 rpm, 20 minutes), the precipitate was subjected to ordinary agarose gel electrophoresis, and a densitometer (CS-9 manufactured by Shimadzu Corporation) was used.
000), it was found that almost 0.5 μg of the entire plasmid DNA had been recovered.

Example 3 (Confirmation of Not Inhibiting Enzymatic Reaction) Linear plasmid DN recovered by centrifugation in Example 2 above
A precipitate (0.5 μg) was dissolved in 25 μl of 10 mM Tris-HCl buffer (pH 8.0, containing 1 mM EDTA). On the other hand, as a control, 0.5 μg of the linear plasmid DNA before electrophoresis was added to the above Tris-HCl buffer (25 μg).
A solution dissolved in 1 was also prepared.

Using 5 μl of each solution, T
4 The linear plasmid DNA was closed by DNA ligase reaction (using a ligation kit manufactured by Takara). Next, the Escherichia coli JM109 strain was subjected to the Hanahan method (Hana
Han, D., J. Mol. Biol., 166, 557 (1983)). The transformed bacteria were spread on an LB agar medium (manufactured by Gibco) plate containing ampicillin (100 μg / ml), cultured at 37 ° C. for 17 hours, and the transformation efficiency was determined from the number of colonies. D recovered from electrophoresis gel for separation and recovery
NA gave transformation efficiencies comparable to control DNA. From the above results, it was confirmed that the sample purified and recovered using the electrophoresis gel for separation and recovery of the present invention did not inhibit the enzyme activity.

[0048]

As described above, the electrophoresis gel for separation and recovery of the present invention can exhibit an aqueous solution (sol) state at a temperature lower than a specific temperature (sol-gel transition temperature) and a hydrogel state at a temperature higher than the specific temperature. It is possible. Therefore, the electrophoresis gel for separation and recovery of the present invention can be made into an aqueous solution at a temperature lower than its sol-gel transition temperature, and thus the gel can be easily formed into a desired shape for electrophoresis. The gelation of the electrophoresis gel for separation and recovery of the present invention is achieved without causing any chemical reaction, and is achieved only by raising the temperature above its sol-gel transition temperature. Therefore, the gelation can be performed extremely simply and quickly.

After the electrophoresis, dissolution of the gel for recovering the sample to be separated can be carried out only by cooling to a temperature lower than the sol-gel transition temperature, so that it is simple and does not induce denaturation of the sample.

In the present invention, a gel material which is soluble in ethyl alcohol can be used, but even in this case, separation of the sample to be separated from the gel material is extremely easy.

Further, since the separation / recovery electrophoresis gel of the present invention can be obtained synthetically, a material free from unknown impurities as found in natural materials can be easily obtained, and Uniformity and stability of performance can be easily assured, and it can be easily assured that the collected sample does not inhibit the enzyme activity.

[0052]

Continuation of the front page (56) References JP-A-4-278451 (JP, A) JP-A-3-296657 (JP, A) JP-A-4-278452 (JP, A) JP-A-5-133937 (JP) JP-A-6-167474 (JP, A) JP-A-5-215717 (JP, A) JP-A-8-503543 (JP, A) JP-A-6-504612 (JP, A) (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 27/447 B01D 57/02 JICST file (JOIS)

Claims (5)

(57) [Claims] Claims: 1. A polymer compound and a polymer compound retained by the polymer compound .
A gel containing at least water; and a sol-gel transition that becomes a sol at a temperature lower than the sol-gel transition temperature and a gel at a temperature higher than the sol-gel transition temperature occurs reversibly. Electrophoresis gel for separation and recovery. Wherein said polymer compound is, Bro having a cloud point
And click, a block copolymer containing other water-soluble block, Bro with該曇point in the block copolymer
The electrophoresis gel for separation and recovery according to claim 1, wherein the electrophoresis gel is a copolymer having a plurality of blocks. 3. The electrophoresis gel for separation and recovery according to claim 1 , wherein the polymer compound is soluble in ethyl alcohol. 4. A polymer compound and the polymer compound retained by the polymer compound.
A gel containing at least water; and a sol-gel
At temperatures lower than the transition temperature, the sol or sol-gel transition temperature
The sol-gel transition, which becomes a gel at higher temperatures, is thermoreversible
Using the separation recovery electrophoresis gel that occurs; sol - as a support of the gel <br/> gel state at a temperature higher than the gel transition temperature, after separation of the material by electrophoresis, the sol the gel - Gel A separation and recovery method, wherein the separated substance is recovered by cooling to a temperature lower than the transition temperature to form a sol state. 5. A polymer compound soluble in ethyl alcohol.
And a gel containing at least water retained thereby.
And at temperatures below the sol-gel transition temperature
Gels at temperatures higher than the sol-gel transition temperature
Electrophoresis for separation and recovery in which Ru-gel transition occurs thermoreversibly
Using gel; sol - as a support of the gel <br/> gel state at a temperature higher than the gel transition temperature, after separation of the material by electrophoresis, by dissolving the gel in ethyl alcohol, separated the A separation and recovery method comprising recovering a substance.