JP2753762B2 - DNA-immobilized microsphere and method for purifying DNA transcription factor using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a DNA-immobilized microsphere used for a protein purification method or the like. More specifically, a DNA strand having a base sequence that specifically binds a carrier made of a resin having an epoxy group to a specific protein and a base sequence that binds to the epoxy group by opening the epoxy group and binds the specific protein in the DNA chain. After adsorbing the protein via the DNA chain to DNA-immobilized microspheres that do not adsorb the protein except for the base sequence portion that specifically binds, and removing other proteins,
The present invention relates to a DNA-immobilized microsphere used in purification of a specific protein that dissociates a DNA chain from the protein.

(Prior Art) One of the pillars of genetic engineering that has made remarkable progress in recent years is the analysis of genes themselves. Among them, analysis of transcriptional regulators in genes leads to elucidation of the mechanism of transcriptional regulation and is important.

Transcription is the process by which RNA is synthesized from DNA, which is a gene, by RNA polymerase, and is divided into transcription initiation, elongation, and termination.

In the transcription initiation stage, RNA polymerase binds to a specific base sequence of DNA called a promoter and initiates transcription. In prokaryotic promoters, called a primobno box, which is located about 10 base pairs upstream from the transcription start site, the nucleotide sequence is slightly different depending on the promoter, but the basic nucleotide sequence is TATAAT.
It is thought to be the site where the polymerase binds.
In addition, certain proteins bind to a specific base sequence called an operator to promote or suppress transcription from a promoter. In eukaryotic cells, the promoter is found 20 to 30 base pairs upstream of the transcription initiation site called the TATA box or Goldberg-Hognes box. Has been suggested to regulate the initiation of transcription of RNA polymerase II, and factors that bind to the TATA box and regulate transcription have also been isolated. The control of the amount of transcription is upstream of the TATA box, and it is thought that each gene has a unique specific base sequence involved in the transcription control. There is also a specific base sequence called an enhancer that increases transcription efficiency. There are three types of eukaryotic RNA polymerases: RNA polymerase II for normal pre-mRNA synthesis, RNA polymerase I for ribosomal RNA synthesis,
RNA polymerase III is involved in the synthesis of 5S and tRNA, and in each case, there is a regulatory gene specific for its transcription.

In the transcription elongation step, in the case of prokaryotic details, there is a sequence called an attenuator as a transcription termination signal present inside the operon, and regulates gene expression.
In the case of animal cells, few regulatory signals corresponding to attenuators have been reported, but they have been suggested to be present in the late gene region of SV40.

In the transcription termination stage, in the case of a prokaryotic cell, there is a nucleotide sequence for terminating transcription called a terminator.
In Escherichia coli, terminators are roughly classified into two types: those that require a protein factor called ρ (ρ-dependent type) and those that do not (ρ-independent type). Also, λN
The terminator is complicatedly controlled by involvement of an anti-transcription termination protein and the like. Research on eukaryotic cells is progressing, but is not clear.

Thus, transcription involves a number of signals on genes. Many of these signals function by interacting with transcription factors, which are proteins. Elucidation and utilization of these transcription mechanisms bring great progress in controlling biological activities and producing useful substances using genetic engineering, and have great significance in the fields of medicine, fermentation, and the like.

Analysis of transcription factors in this important gene requires separation and purification of proteins that serve as regulators. However, such a transcription factor protein is very small and difficult to purify. Transcription factor proteins that directly bind to genes can be separated and analyzed using affinity chromatography with the DNA strand containing the binding site as the ligand.
Purification has been performed, and gel particles such as cellulose and agarose have been used. However, these commercially available gels are
In most cases, the particle size is several tens of μm or more, the total area of the particle surface is small, and the particle size distribution is wide,
When used for the above applications, it cannot be said that they have sufficient performance in terms of efficiency, reproducibility, and the like. Furthermore, when packed into a column, these gel particles contain water several tens of times or more, so from the viewpoint of the strength of the gel particles, it is impossible to flow the solution through the column at high pressure, and the efficiency is high. Is bad.

In addition, the present inventors have used a synthetic polymer particle having a base sequence to which a protein, which is a transcription control factor, is chemically bonded to a synthetic polymer particle having no nonspecific adsorption property as a carrier, It has been found that the protein which is the transcription control factor can be obtained easily and in good yield.
It was announced at the 37th Symposium on Polymers in 2000.

(Problems to be Solved by the Invention) As a result of intensive studies, the present inventors have found that a DNA strand having a base sequence that specifically binds a carrier composed of a resin having an epoxy group to a specific protein is formed on the epoxy chain. DN that binds by opening the group and does not adsorb proteins other than the base sequence portion that specifically binds the specific protein in the DNA chain
By using the A-immobilized microspheres, they have found that the specific protein can be obtained more easily and with higher yield, and have completed the present invention.

(Means for Solving the Problems) According to the present invention, as a first invention, a base sequence that specifically binds a specific protein to a carrier having a particle size of 50 μm or less and made of a resin having an epoxy group on the surface is provided. A DNA-immobilized microsphere which does not adsorb proteins except for a base sequence portion which has a DNA chain bound by opening the epoxy group and specifically binds to a specific protein in the DNA chain is provided. After adsorbing the protein to the DNA-immobilized microspheres of the first invention via the DNA chain and removing other proteins, the protein and DNA
Methods are provided for the purification of certain proteins that cause chain dissociation.

In the purification method using the DNA-immobilized microspheres of the present invention, when a plurality of proteins bind as subunits and function as one factor, a plurality of proteins may be obtained. May include proteins that do not bind directly. In the present invention, such a protein is included in a protein that binds to a DNA chain as long as it binds to another protein and functions as one factor. In addition, as long as only proteins that bind to each other and function as one factor are obtained, even if a plurality of types of proteins are obtained, it is referred to as purification in the present invention.

The carrier in the present invention has an epoxy group on the surface, and after the DNA chain is bonded by opening the epoxy group, the protein is not substantially adsorbed on the surface of the carrier by physical adsorption or the like. If the surface satisfies these conditions, the structure of the carrier is not limited, and the entire carrier may be composed of only one type of polymer or copolymer, or may have a core-shell structure .

When the carrier in the present invention is packed in a column chromatography for protein purification and used, it is required to have strength, specific gravity and size corresponding thereto.

For the purpose of use for purification, those having many DNA chains bound per unit volume are preferable. For that reason, the smaller the particle size of the spherical carrier, the better. This is because when the particle size is small, the surface area per unit volume increases, and the number of sites to which DNA chains can bind increases. A particle size of 50 μm or less, preferably 20 μm or less is effective.

However, on the other hand, when considering the use for affinity chromatography, if the particle size is 3 μm or less, it is difficult to pack and maintain the column. This is because there is no material that satisfies the conditions required for the material for preventing the carrier from flowing out of the column and prevents particles having a particle size of 3 μm or less from flowing out. However, even if the particle size is 3 μm or less, it can be used for protein purification as long as it can be used for separation / purification by a batch method in which a protein is adsorbed on a DNA chain and then separated from a sample.

In the case of packing in a column, if the specific gravity is too small, packing is difficult.

On the other hand, when using a batch-type protein purification method,
Since it is preferable that the DNA-fixed microspheres and supernatant can be easily separated by centrifugation and can be easily dispersed in the solution, determine the specific gravity of the carrier based on the specific gravity of the solution used as a solvent in protein purification such as a buffer. Is preferable. When the specific gravity of the resin used as the carrier surface is not preferable, the specific gravity of the entire carrier may be preferable, such as a core-shell structure in which a core having an appropriate specific gravity is coated with the resin used as the carrier surface. .

When the carrier has a core-shell structure and the core substance is a substance that adsorbs proteins, the surface must be completely coated, and the surface may be peeled off during the purification operation. Sufficient strength is required so as not to be.

In general, when the particle surface is a hydrophobic surface, various proteins are likely to cause non-specific adsorption. Therefore, in the present invention, it is essential that the surface of the carrier after the binding of the DNA chain is not hydrophobic. Therefore, the resin on the carrier surface of the DNA-immobilized microsphere of the present invention is exemplified by a polymer of an epoxy group-containing (meth) acrylate such as glycidyl (meth) acrylate. The resin on the carrier surface has an epoxy group on the surface of the carrier, as long as the DNA chain satisfies the condition that the protein is not adsorbed to the carrier surface by physical adsorption or the like after the DNA chain is opened and bonded. It may be a homopolymer or a copolymer. For example, a copolymer of an epoxy group-containing (meth) acrylate and a monomer of a hydrophilic polymer compound may be used. Examples of the hydrophilic polymer compound include ethylene oxide-containing (meth) acrylates such as ethylene glycol (meth) acrylate and triethylene glycol (meth) acrylate, hydroxymethyl (meth) acrylate, and (meth) acrylic acid. Hydroxy group-containing (meth) acrylates such as hydroxypropyl, alkyl (meth) acrylates such as methyl (meth) acrylate and ethyl (meth) acrylate, acrylamide, methacrylamide, diacetacrylamide, N-hydroxyethylacrylamide, etc. Monoethylenically unsaturated amide monomer, acrylonitrile, methacrylonitrile and other ethylenically unsaturated nitrile compounds. Even if it is not a hydrophilic monomer, any monomer can be used as a copolymer with an epoxy group-containing (meth) acrylate monomer, as long as it does not bring nonspecific adsorption of protein onto the carrier surface. In the present invention, a polymer of glycidyl methacrylate (hereinafter, referred to as GMA) is particularly preferred from the viewpoint of preventing protein adsorption.

The method for producing the carrier of the DNA-immobilized microspheres of the present invention comprises:
The surface of the manufactured carrier has an epoxy group, and DNA
There is no particular limitation as long as the protein is not adsorbed on the surface of the carrier by physical adsorption or the like after the chain is bonded by opening the epoxy group. However, most of those widely used as polymerization initiators cause many of the epoxy groups to be ring-opened when a monomer containing an epoxy group is polymerized. Absent. In the present invention, in order to produce the resin on the surface of the carrier, it is necessary to use a polymerization initiator for polymerizing a monomer containing an epoxy group without opening the epoxy group. As such a polymerization initiator, for example,
There is a water-soluble azo polymerization initiator. Examples of the water-soluble azo polymerization initiator include 2,2'-azobis (2-amidinopropane) dihydrochloride (hereinafter, referred to as V50) and 4,4'-
Azobis (4-cyanovaleric acid), 2,2'-azobis (N,
N'-dimethyleneisobutylamidine) dihydrochloride, 2,2'-azobis (N, N'-dimethyleneisobutylamidine), 2,2'-azobis {2-methyl-N- [1,1
-Bis (hydroxymethyl) -2-hydroxyethyl]
Propionamide, 2,2'-azobis {2-methyl-
N- [1,1-bis (hydroxymethyl) -ethyl] propionamide {, 2,2'-azobis [2-methyl-N-
(2-hydroxymethyl) propionamide], 2,2 '
-Azobis (isobutylamide) dihydrate and the like.

These water-soluble azo polymerization initiators can be used in a soap-free emulsion polymerization method without using a surfactant.
Carriers produced by this method are preferred because they do not use surfactants that can contaminate the carrier surface and cause non-specific adsorption of proteins.

In the polymerization of the resin on the carrier surface, a crosslinking agent or the like may be used as necessary. The crosslinker has a small effect on the carrier surface. For this reason, a hydrophobic cross-linking agent can be used, but it is preferable to use a hydrophilic cross-linking agent that is unlikely to cause nonspecific adsorption of proteins. As the hydrophilic crosslinking agent, ethylene glycol dimethacrylate, tetramethylene glycol dimethacrylate,
Examples thereof include divinylbenzene.

Although GMA can be polymerized by a soap-free emulsion polymerization method using a water-soluble azo-based polymerization agent, conditions are difficult, and it is difficult to obtain particles of stable quality when used for purification. When GMA is used as the surface of the carrier, the carrier preferably has a core-shell structure.

The DNA strand immobilized on the carrier in the present invention is 2
It is a main chain. If it is single-stranded, a part of the DNA chain may be involved in the immobilization during immobilization on the particles, and non-specific adsorption of the protein is likely to occur.

This DNA strand has a base sequence that can specifically bind to a specific protein.
It is selected from proteins that bind to the DNA chain, and a specific base sequence is selected according to the protein. In order to improve the purification efficiency, it is preferable that a plurality of specific base sequences binding to a specific protein are contained in one DNA chain.

As a protein that specifically binds to a specific base sequence, there is a transcription control factor protein and the like.

The immobilization of the DNA chain on the carrier surface is based on a covalent bond that opens the epoxy group, and the amino group of the single strand at both ends of the DNA chain is reacted with the epoxy group on the carrier surface to form a DN.
Fix A chain.

After fixing the DNA strand, it is preferable to eliminate the reactivity of the epoxy group. This is because if an epoxy group having reactivity remains, the amino group of the protein and the epoxy group may react. As a method for eliminating the reactivity of the epoxy group, for example, there is a method of treating with ethanolamine hydrochloride.

The protein is purified using this DNA-immobilized microsphere, but the method of adsorbing the target protein and removing other proteins, and then dissociating the specific protein from the DNA chain is not particularly limited. Affinity chromatography using a column or a batch type may be used.

Since these particles are harder than gels and do not contain water, when packed in a column, it is possible to flow the solution at a high pressure, and purification can be performed efficiently. Also, conventional column chromatography using gels requires a large amount of buffer, resulting in dilution of the purified protein. On the other hand, purification using these particles not only has a low dilution ratio in the case of column chromatography, but also in a batch method, such as sedimentation of the particles, is simple and does not dilute the purified protein. Obtainable. This is a great advantage in the purification of a trace protein such as a transcription factor protein.

Further, in the conventional purification of a transcription control factor, when a nuclear extract of a cell is used, a pretreatment such as using a column of heparin sepharose was required. However, when the DNA-immobilized microsphere of the present invention is used, such a pretreatment is unnecessary. In the case where multiple types of proteins bind to each other and function as one factor,
Some proteins constituting one factor may be removed by pretreatment, and such proteins could not be obtained only by a conventional purification method requiring pretreatment. In the purification method using DNA-immobilized microspheres, it can be obtained simultaneously with other proteins.

The DNA-immobilized microspheres of the present invention can be used not only for protein purification but also for detection of specific proteins that specifically bind to immobilized DNA chains.

(Effect of the Invention) Thus, the DNA-immobilized microspheres of the present invention have a large number of immobilizable DNA chains, and according to the protein purification method using the same, specific proteins that specifically bind to specific nucleotide sequences, particularly transcription control factor proteins However, compared with the conventional method, separation and purification can be performed more efficiently.

(Example) Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 1.8 g of monomer GMA and 120 g of monomer / styrene in 120 g of distilled water
Add 1.2g, divinylbenzene 0.04g, rotation speed 200rpm
The mixture was purged with nitrogen at 70 ° C. for 30 minutes while stirring with a three-one motor. To this was added 500.06 g of V dissolved in 10 g of distilled water and polymerized at 70 ° C. for 2 hours. 0.3 g of a monomer GMA was added, and the mixture was further polymerized for 22 hours. After the polymerization, the reaction solution was dispensed into four 50 ml centrifuge tubes, centrifuged at 13000 rpm for 10 minutes at 20 ° C., and the supernatant was removed. Then, distilled water was added, re-dispersed and washed three times. The particles thus obtained (hereinafter, referred to as
Coated particles) has an average particle diameter of 0.173 μm and a specific gravity of
Since it is as large as 1.285, it has the advantage of easy sedimentation and easy separation and recovery.

COMPARATIVE EXAMPLE 1 3.0 g of monomer GMA and 0.2 g of divinylbenzene in 120 ml of distilled water.
After adding 04 g, the mixture was purged with nitrogen at 70 ° C. for 30 minutes while stirring with a three-one motor at a rotation speed of 200 rpm. To this, potassium persulfate (0.06 g) dissolved in 10 g of distilled water was added as a polymerization initiator, and the mixture was polymerized at 70 ° C. for 24 hours. After polymerization, add 50
After dispensing into four ml centrifuge tubes and centrifuging at 20 ° C. and 13,000 rpm for 10 minutes to remove the supernatant, distilled water was added, re-dispersed and washed three times. The particles thus obtained (hereinafter referred to as GMA particles) had an average particle diameter of 0.136 μm and a specific gravity of about 1.2.

Example 2 19 bp oligodeoxynucleotide having a base sequence recognized by transcription control protein E4TF3 Was prepared using a synthesizer.

This oligodeoxynucleoside has γ- ³² P-ATP,
ATP and kinase were added, the reaction was carried out at 37 ° C. for 1 hour, the 5 ′ end was phosphorylated, and annealed / ligase-ligated to prepare a DNA chain of about 300 bp having a protruding 5 ′ end.

Example 3 2.5 mg of coated particles were added to a 10 mM potassium phosphate buffer (pH
8.0) After dispersing in 500 µl, centrifuging the supernatant, removing the supernatant, redispersing in 200 µl of 10 mM potassium phosphate buffer (pH 8.0) to which 30 µg of the DNA strand obtained in Example 4 was added.
Reaction was performed at 24 ° C. for 24 hours. After the reaction, the supernatant was removed by centrifugation, washed twice with a 2.5 M potassium chloride solution, and the radiation count of the pellet and each washing solution was measured. 1M ethanolamine hydrochloride (pH 8.0)
ml was added and left at room temperature for 24 hours to mask unreacted epoxy groups.

The prepared DNA-immobilized microspheres were mixed with a stock buffer (0.3 M sodium chloride, 1 mM ethylenediaminetetraacetic acid,
0.02% sodium azide, 10 mM Tris-HCl (pH 8.0)
And then dispersed in 0.2 ml of the same buffer solution at 4 ° C.
Saved in. The amount of DNA bound was calculated from the radiation count of the system after the reaction. From this result, DNA bound to coated particles
The amount was calculated to be 11.2 μg.

Comparative Example 2 The DNA chain prepared in Example 2 was poly-GMA by the cyanogen bromide method.
Bound to the particles. The reaction conditions were determined in advance,
The poly-GMA particles prepared in Comparative Example 1 were bound at a reaction temperature of 2 ° C., pH 11 to 12, and cyanogen bromide / particle = 3.0 g / 240 mg, which can bind the DNA chain with the highest efficiency. After collecting the poly-GMA particles with the fixed DNA chains by centrifugation, the remaining DNA chains that did not bind to the particles in the supernatant were quantified from the ³² P radiation dose, and DN
As a result of calculating the fixed amount of the A chain, for 12 mg of poly-GMA particles
It was found that 10 μg of the DNA chain was fixed.

Even if the resin on the surface of the carrier is a polymer composed of the same monomer, the carrier with the resin having the epoxy group on the surface can fix a larger number of DNA chains than the carrier with the resin without the epoxy group on the surface. all right.

Example 4 20 μl of a stock buffer in which the DNA-immobilized microspheres obtained in Example 3 were dispersed (the amount of DNA-immobilized microspheres was about
0.25mg), put in a 1.5ml Eppendorf tube,
Once with 100 μl of distilled water, buffer for particle washing (50 mM Tris-HCl (pH 0.8), 1 mM ethylenediaminetetraacetic acid, 0.1 MDT
(T, 20% glycerol, 0.1% pn-octylphenyl ether)).

After washing HeLa cells with PBS containing 0.5 mM MgCl ₂ and 1 mM DTT, a buffer for cell membrane breakage (10 mM HEPES (pH 7.9), 10 mM K
Cl, 1.5 mM MgCl ₂ , 0.5 mM DTT), homogenize, centrifuge to remove the supernatant, and furthermore, a nuclear protein extraction buffer (20 mM HEPES (pH 7.9), 25% glycerol, 0.42
M NaCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM
DTT), centrifuged, and the supernatant was dialyzed to obtain a crude nuclear extract having a protein concentration of 8 mg / ml.

Add 50 μl of the crude nuclear extract to 10 μl of poly [dI-dC] and 10% p
After adding 0.5 μl of -n-octylphenyl ether and stirring, 1
Let stand for 5 minutes, add to the washed DNA-fixed microspheres,
After leaving still for 30 minutes, E4TF3 was bound to the DNA strand of the DNA-immobilized microsphere. The mixed solution such as the crude nuclear extract and the DNA-fixed microspheres was centrifuged in a 1.5 ml Eppendorf tube, the supernatant was separated, and the pellet was washed three times with a particle washing buffer. Then, the pellet is dissolved in an elution buffer (50
mM trihydrochloride (pH 8.0), 1 mM ethylenediaminetetraacetic acid, 1.
0 M potassium chloride, 1 mM DDT, 20% glycerol, 0.1% p
-N-octylphenyl ether) was washed three times with 20 µl to purify E4TF3. By SDS-PAGE, it was confirmed that bands of six proteins of E4TF3 were very selectively separated and purified.

Comparative Example 3 20 μl of a stock buffer in which the DNA-immobilized microspheres obtained in Example 3 were dispersed (the amount of DNA-immobilized microspheres was about
Purification of E4TF3 in the same manner as in Example 6 except that instead of using 0.25 mg), 20 μl of a stock buffer in which the DNA-immobilized microspheres obtained in Comparative Example 2 were dispersed (the amount of DNA-immobilized microspheres was about 0.25 mg). Tried. By SDS-PAGE, E4
Six protein bands of TF3 were confirmed, but at least five or more other proteins were detected.

The DNA-immobilized microspheres of the present invention were found to be more selective in protein purification than conventional DNA-immobilized microspheres.

Claims (6)

(57) [Claims] 1. A DNA strand having a base sequence which specifically binds a specific protein to a carrier having a particle size of 50 μm or less, which is made of a resin having an epoxy group on the surface, is bonded by opening the epoxy group. DNA-immobilized microspheres that do not adsorb proteins other than the base sequence portion that specifically binds to a specific protein. 2. The DNA-immobilized microsphere according to claim 1, wherein the specific protein is a transcription factor. 3. The DNA-immobilized microsphere according to claim 1, wherein the surface is made of polyglycidyl methacrylate. 4. A DNA strand having a base sequence which specifically binds a specific protein to a carrier having a particle size of 50 μm or less, which is made of a resin having an epoxy group on the surface, is bonded by opening the epoxy group. After adsorbing the protein via the DNA chain to DNA-immobilized microspheres that do not adsorb the protein except for the base sequence portion that specifically binds the specific protein, and after removing other proteins, the protein A method for purifying specific proteins that dissociates DNA strands. 5. The method for purifying a specific protein according to claim 4, wherein the specific protein is a transcription control factor. 6. The method for purifying a specific protein according to claim 4, wherein said surface is made of polyglycidyl methacrylate.