JPH0446193A - Dna immobilized microsphere and purification of dna transcription control factor using the same microsphere - Google Patents

Abstract

PURPOSE:To obtain DNA immobilized microspheres capable of being purified from a specific protein readily and in high yield, comprising a specific DNA chain bonded to a carrier composed of a resin containing epoxy group on the surface by covalent bond to subject the epoxy group to ring opening. CONSTITUTION:DNA immobilized microspheres comprising a DNA chain having a base sequence to specifically bond a specific protein, bonded to a carrier composed of a resin containing epoxy group on the surface by subjecting the epoxy group to ring opening and not adsorbing protein except the base sequence part. An epoxy group-containing (meth)acrylic ester (co)polymer may be cited as the resin to form the carrier surface and polyglycidyl methacrylate prepared by soap-free emulsion polymerization using especially a water-soluble azo-based polymerizing agent is preferable. A DNA transcription control factor may be cited as the protein to be purified by using DNA immobilized microspheres.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to DNA immobilized microspheres used in protein purification methods and the like. More specifically, a carrier made of a resin having an epoxy group on its surface and DNAIN, which has a base sequence that specifically binds a specific protein, open the ring of the epoxy group and bond to the DNA! The DNA is attached to DNA immobilized microspheres that do not adsorb proteins except for the base sequence part that specifically binds a specific protein.
The present invention relates to DNA immobilized microspheres used in the purification of a specific protein, which adsorbs the protein via AII, removes other proteins, and then dissociates the protein and DNA strand.

(Conventional technology) One of the pillars of genetic engineering, which has made remarkable progress in recent years, is the analysis of genes themselves. Among these, analysis of transcriptional control factors in genes is important because it leads to elucidation of the mechanism of transcriptional control.

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

At the transcription M# stage, RNA polymerase binds to a specific DNA base sequence called a promoter and initiates transcription. In prokaryotic cell promoters, the nucleotide sequence called the pribnow box, which exists 10 base pairs upstream from the transcription start site, differs slightly depending on the promoter, but the basic nucleotide sequence is TATAAT, is the nucleotide sequence that RNA polymerase actually binds to. It is thought that this is the part where Additionally, certain proteins bind to specific base sequences called operators and promote transcription from promoters. or suppress.

In eukaryotic cells, the TATA box or boldbarb
20 upstream from the transcription start site called the Hogness box
It has been suggested that a nucleotide sequence found at a position of ~30 base pairs (which varies slightly depending on the promoter, but basically represents) regulates transcription initiation of RNA polymerase H, and binds to the TATA box. Factors that regulate transcription have also been isolated. Control of transcription amount is TATA
It is thought that there are unique and specific base sequences located upstream of the box and involved in the transcriptional control of each gene. Furthermore, there are specific base sequences called enhancers that increase transcription efficiency. There are three types of RNA polymerase in eukaryotic cells: RNA polymerase ■ is involved in the synthesis of normal mRNA precursors, RNA polymerase ■ is involved in the synthesis of liposomal RNA, and RNA polymerase ■ is involved in the synthesis of 5S and tRNA. In each case, there are regulatory genes specific for that transcription.

At the transcription elongation stage, in the case of IM nuclear vacuoles, there is a sequence called an attenuator as a transcription termination signal that exists inside the operon and regulates gene expression.

Although there are few reports of regulatory signals corresponding to attenuators in animal cells, it has been suggested that they exist within the late gene region of SV40.

At the transcription termination stage, in the case of -m nuclear lateral vesicles, there is a base sequence called a terminator that instructs transcription termination. In E. coli, there are two types of terminators: those that require a protein factor called ρ (ρ-dependent type) and those that do not (ρ-independent type). In addition, the terminator is complicatedly tangled due to the involvement of λN anti-transcription termination protein and the like. Research on eukaryotic cells is progressing, but the results remain unclear.

In this way, many genetic signals are involved in transcription. Many of these signals function through interaction with transcription control factors, which are proteins. The elucidation and utilization of these transcription mechanisms will bring great progress in the control of life activities and the production of useful products using genetic engineering, and will have great significance in fields such as medicine and fermentation.

In order to analyze the transcriptional control factor of this important gene, it is necessary to isolate and purify the protein that serves as the control factor. However, such transcriptional sq control factor proteins are present in minute amounts, making it difficult to am.

Transcription control factor proteins that directly bind to genes have been separated and produced using affinity chromatography using a DNA strand containing a binding site as a ligand, and gel particles such as cellulose or agarose have been used. However, most of these commercially available gels have a particle size of several tens of nanometers or more, and the total surface area of the particles is small.
Furthermore, since the particle size distribution is wide, it cannot be said that it has sufficient performance in terms of efficiency, reproducibility, etc. when used in the above applications. Furthermore, when packed in a column, these gel particles contain several tens of times more water, so considering the strength of the gel particles, it is impossible to flow the solution into the column at high pressure, which reduces efficiency. It is a bad thing.

In addition, the inventors discovered that using synthetic polymer particles that do not have non-specific adsorption properties and chemically bonded synthetic NA chains with a base sequence that specifically binds a protein, which is a transcription control factor, as a carrier. , found that the protein which is the transcriptional control factor can be obtained easily and in good yield, and 19
Presented at the 37th Polymer Symposium in 1988.

(Problems to be Solved by the Present Invention) As a result of a series of researches, the present inventors discovered that a DNA strand having a base sequence that specifically binds a specific protein to a carrier made of a resin having an epoxy group on the surface. By using DNA immobilized microspheres that bind by ring-opening epoxy groups and do not adsorb proteins other than the base sequence region that specifically binds a specific protein in the DNA chain, the specific protein can be more easily absorbed. The present inventors have found that they can be obtained in higher yields, and have completed the present invention.

(Means for Solving the Problems) Thus, according to the present invention, as an invention of fJl, a carrier made of a resin whose surface has an epoxy group and a DNA chain having a base sequence that specifically binds a specific protein are bonded to the epoxy group. A second invention provides a DNA-immobilized microsphere that binds by ring-opening groups and does not adsorb proteins other than the base sequence portion that specifically binds a specific protein in DNA II. adsorbing the protein to DNA immobilized microspheres via MDNll;
After removing other proteins, the protein and DNA!
Provided are methods for purifying specific proteins that dissociate.

In the purification method using DNA immobilized microspheres of the present invention, when multiple proteins are combined as subunits and function as one factor, multiple types of proteins may be obtained. may include proteins that do not bind directly. Since such proteins function as a factor by binding with other proteins, they are included in the proteins that bind to DNA chains in the present invention. Further, as long as only proteins that substantially bind to each other and function as a single factor are obtained, even if several types of proteins are obtained, the term "purification" is used in the present invention.

The carrier in the present invention has an epoxy group on its surface, and after DNAtIjl rings-opens and bonds the epoxy group, proteins are not substantially adsorbed to the carrier surface by physical adsorption or the like.

As long as the surface satisfies these conditions, there are no restrictions on the structure of the carrier, and it does not matter if the entire carrier is made of only one type of polymer or copolymer or has a core-shell structure. .

When the carrier of the present invention is used in column chromatography for protein purification, it is required to have strength, specific gravity, and size appropriate for the purpose.

For the purpose of purification, it is preferable that a large amount of DNA II be bound per unit volume. For that reason,
The smaller the particle size of the spherical carrier, the more preferable it is. This is because the smaller the particle size, the larger the surface area per unit volume, which increases the number of sites to which DNA chains can bind. Particle size is 50I
115 or less, preferably 20 [phases] or less, is effective.

However, when considering use in affinity chromatography, if the particle size is 3 (7) or less, it is difficult to fill and retain the particles in a column. This is because there is no material that satisfies the requirements for a material to prevent carriers from flowing out of the column and can prevent particles with a particle size of 3 or less from flowing out. However, even if the particle size is 3gn or less, DNA! If it can be used for separation and purification in a batch method in which proteins are adsorbed onto a sample and then separated from a sample, it can be used for protein purification.

Furthermore, when filling a column, if the specific gravity is too low, it is difficult to fill the column.

On the other hand, when using a batch-type protein purification method, it is preferable that the DNA-immobilized microspheres and the supernatant can be easily separated by centrifugation, and that they can be easily dispersed in the liquid. It is preferable to determine the specific gravity of the carrier based on the specific gravity of the liquid.

If the specific gravity of the resin used as the carrier surface is unfavorable, the specific gravity of the carrier as a whole may be made preferable, such as by using a core-shell structure carrier in which a core with an appropriate specific gravity is coated with the resin used as the carrier surface. .

If 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 must not peel off during the purification process. Sufficient strength is required to ensure that no damage occurs.

- When the particle surface is numerically water permeable, various proteins are likely to cause non-specific adsorption.

Therefore, in the present invention, it is essential that the carrier surface of the edge to which DNAJI is bound is not hydrophobic.

Therefore, the resin on the carrier surface of the DNA immobilized microspheres of the present invention is exemplified by a polymer of epoxy group-containing (meth)acrylic acid ester such as glycidyl (meth)acrylate. The resin on the surface of the carrier can be It may be a homopolymer or a copolymer.

For example, it may be a copolymer of an epoxy group-containing (meth)acrylic acid ester and a hydrophilic polymer compound monomer. Examples of hydrophilic polymers include ethylene oxide-containing (meth)acrylic acid esters such as ethylene glycol (meth)acrylate and triethylene glycol (meth)acrylate;
Hydroxy group-containing (meth)acrylic acid esters such as acrylic t-hydroxymethyl, hydroxypropyl (meth)acrylate, methyl (meth)acrylate, ethyl (
Alkyl (meth)acrylic esters such as meth)acrylate, monoethylenically unsaturated amide monomers such as acrylamide, methacrylamide, diacetoacrylamide, N-hydroxyethyl acrylamide, ethylenically unsaturated nitrile compounds such as acrylonitrile, methacrylaterile, etc. can be exemplified. Even if it is not a hydrophilic monomer, as long as it is a copolymer with an epoxy group-containing (meth)acrylic acid ester monomer and does not cause non-specific adsorption of proteins to the carrier surface,
Can be used. In the present invention, from the viewpoint of preventing protein adsorption, a polymer of glycidyl methacrylate (hereinafter referred to as GMA) is particularly preferred.

The method for producing the carrier of DNA immobilized microspheres of the present invention is as follows:
The surface of the prepared carrier has an epoxy group, and D
There is no particular limitation as long as the protein does not adsorb to the surface of the carrier by physical adsorption or the like after NA11 rings-opens and bonds the epoxy group. However, most of the widely used polymerization initiators open many of the epoxy groups when polymerizing monomers containing epoxy groups, so they cannot be used in the preparation of the support of the present invention. Undesirable. In the present invention, in order to produce the resin on the surface of the carrier, it is necessary to use a polymerization initiator that polymerizes a monomer containing an epoxy group without ring-opening the epoxy group. Such polymerization initiators include:
For example, there are water-soluble azo polymerization initiators. Examples of water-soluble azo polymerization initiators include 2,2'-azobis(2-amidinopropane) dihydrochloride (hereinafter referred to as v50), 4°4'-azobis(4-cyanovaleric acid), 2,2
'-azobis(N,N'-dimethyleneisobutyramidine) dihydrochloride, 2,2'-azobis(N,N
'-dimethyleneisobutyramidine), 2,2'-azobis(2-methyl--[1,1-bis(hydroxymethyl)-2-hydroxyethyl]propionamide),
2,2'-azobis(2-methyl ~-[1,■bis(
hydroxymethyl)~ethyl]propionamide), 2
, 2'-azobis[2-methyl-N(2-hydroxymethyl)propionamitoco, 2,2'-azobis(isobutyramide) dihydrate and the like.

These water-soluble azo polymerization initiators can be used in a soap-free emulsion polymerization method that does not use a surfactant. A carrier made of W using this method is preferable because it does not use a surfactant that can contaminate the carrier surface and cause non-specific adsorption of proteins.

In the polymerization of the resin on the surface of the carrier, a crosslinking agent or the like may be used as necessary. The crosslinking agent has little effect on the carrier surface. Therefore, although a hydrophobic cross-linking agent can be used, it is preferable to use a hydrophilic cross-linking agent that is less likely to cause non-specific adsorption of proteins. Examples of the hydrophilic crosslinking agent include ethylene glycol dimethacrylate, tetramethylene glycol dimethacrylate, and divinylbenzene.

Although it is possible to polymerize GMA using a soap-free emulsion polymerization method using a water-soluble azo polymerization agent, there are no conditions and it is difficult to obtain particles of stable quality for use in purification. . When GMA is used as the surface of a carrier, it is preferable to use a carrier having a core-shell structure.

In the present invention, the DNAJ to be immobilized on the carrier is 2
It is the main chain. If it is single-stranded, the DNA will be immobilized on the particle.
There is a possibility that JI arrest is involved in immobilization, and nonspecific adsorption of proteins is likely to occur.

This DNA II has a base sequence that can specifically bind to a specific protein, and the target protein is DNA! A specific base sequence is selected depending on the protein that binds to the protein. In order to improve the efficiency of purification, it is preferable that one DNAII contains multiple specific base sequences that bind to a specific protein.1/X0 As a protein that specifically binds to a specific base sequence Examples include transcription control factor proteins.

The DNA strands are immobilized on the carrier surface by covalent bonds that ring-open the epoxy groups, and DNA II is immobilized by reacting the amino groups of the single-stranded portions at both ends of DNA11 with the epoxy groups on the carrier surface.

Furthermore, DNA! After fixing, it is preferable to eliminate the reactivity of the epoxy group. This is because if a reactive epoxy group remains, there is a possibility that the amino group of the protein and the epoxy group will react. As a method for eliminating the reactivity of epoxy groups, there is, for example, a method of treatment with @ acid ethanolamine.

Using these DNA-fixed microspheres, proteins [1! However, the method of adsorbing the target protein and removing other proteins and then dissociating the specific protein and DNAJI is not particularly limited. Affinity chromatography using a column or batch method may be used.

These particles are harder than gel and do not contain water, so when packed into a column, it is possible to flow the solution under high pressure, allowing efficient purification. In addition, in conventional column chromatography using gel, a large amount of w
I buffer is required, resulting in dilution of 1 ftll of protein. On the other hand, in purification using these particles, not only is the dilution ratio low in column chromatography, but also in batch methods, ff! i Simply and undiluted-
#II protein can be obtained. This is a great advantage in purifying trace amounts of proteins such as transcriptional regulatory factor proteins.

Furthermore, in conventional purification of transcription control factors, when cell nuclear oil extracts are used, pretreatment such as using a heparin Sepharose column is required. However, when using the DNA-immobilized microspheres of the present invention, such pretreatment is not necessary.

In cases where multiple types of proteins bind to each other and function as a single factor, some of the proteins that make up a single factor may be removed by pretreatment; could not be obtained using only conventional purification methods that require pretreatment, but can be obtained simultaneously with other proteins using the purification method using DNA-immobilized microspheres of the present invention.

In addition, the DNA-immobilized microspheres of the present invention can be used not only for the detection of 1# proteins but also for the detection of specific proteins that specifically bind to immobilized DNA strands.

(Effects of the Invention) Thus, the DNA-immobilized microspheres of the present invention can immobilize a large amount of DNA, and according to the tanhaku WIl method using this, a specific protein, especially a transcription control factor protein, that specifically polymerizes with a specific base sequence can be immobilized. , it can be separated and purified more efficiently than conventional methods.

(Example) The present invention will be described in more detail with reference to Examples below.

120 g of distilled water, 1.8 g of monomer GMA, 1.2 g of monomer styrene - 0.04 g of divinylbenzene
was added, and the mixture was replaced with nitrogen at 70° C. for 30 minutes while stirring with a three-one motor at a rotational speed of 200 rpm. Add 08g of V2O3 dissolved in 10g of distilled water to this, and add 70g of V2O3.
Polymerization was carried out at ℃ for 2 hours. 3 g of monomer GMA O was added, and polymerization was further carried out for 22 hours. After polymerization, the reaction solution was adjusted to 5M
Dispense into 4 centrifuge tubes and incubate at 20℃, 13,000 rpm+m.
After centrifuging for 0 minutes to remove the supernatant, distilled water was added, redispersion, and washing were performed three times. The particles thus obtained (hereinafter referred to as coated particles) have an average particle diameter (
L173μ east specific gravity is 1. ．． Because it is large (285), it has the advantage of being easy to settle and being easily separated and recovered.

3.0 g of monomer GMA and 0.04 g of divinylbenzene were added to 120 g of distilled water, and the mixture was replaced with nitrogen at 70° C. for 30 minutes while stirring with a three-one motor at a rotational speed of 200 rpm. Potassium persulfate (
Dissolve 6g of LO in 10g of distilled water, add and heat to 70°C12
Polymerization took 4 hours. After polymerization, transfer the reaction solution to a 50-centrifuge tube 4
After removing the supernatant by centrifugation at 13,000 rpm for 10 minutes, distilled water was added, redispersion, and washing were performed three times.The particles thus obtained (hereinafter referred to as GMA particles) ), the average particle diameter is (L13
The specific gravity was approximately 1.2.

Soganka 1 Oligodeoxynucleotide 3'-^A of 19b, which has a base sequence recognized by transcriptional tone protein E4TF3
GTGACGTAAC: GTG to GGG-5'5''-C
CCCCTTCACTGCATTGCA-3' was synthesized using a synthesizer.

This oligodeoxynucleoside contains γ-32pATR.
, ATP, and kinase were added and reacted at 37°C for 1 hour.
The 5' end was phosphorylated and ligated by annealing/ligase to create an approximately 300 bp DNA strand with an overhang at the 5' end.

! After dispersing the coated particles of Gansoyu 2.5 Otori in 500 J of 10 IN potassium phosphate buffer (pH 8,0), centrifugation was performed and the supernatant was removed to obtain the DNA obtained in Example 4. 30μs
lo-N potassium phosphate buffer solution (pH 8,0)
After redispersing in 2004, the mixture was reacted at 50° C. for 24 hours.

After the reaction, the pellet was centrifuged, the supernatant was removed, and the pellet was washed twice with 2.5M potassium chloride solution, and the radiation counts of the pellet and each washing solution were measured. After washing, add 1 M@Ill ethanolamine (pH 8,0) to the DNA immobilized microspheres.
l- was added and left at room temperature for 24 hours to mask unreacted epoxy groups.

The prepared DNA-immobilized microspheres were mixed with stock buffer (
Washed three times with 0.3M sodium chloride, Id ethylenedicyanetetraacetic acid, 0.02% sodium azide, 10mM Tris salt 1t (pH 8-0)), and added the same buffer solution 0.
-2- and stored at 4°C. The amount of DNA bound was calculated from the radiation count of the system after the reaction.

From this result, the amount of DNA bound to the coated particles is 11.
It was calculated to be 2μ8.

11th base The DNAJ prepared in Example 2 was converted to polyG using the cyanogen bromide method.
bound to MA particles. As a result of a preliminary study, the reaction conditions were as follows: a reaction temperature of 2°C, PHII ~ 12, which allows DNA1i to bind with the highest efficiency to the polyGMA particles prepared in Comparative Example 1.
, cyanogen bromide/particle=3.0g/240mg. After recovering the polyGMA particles with immobilized DNA strands by centrifugation, the D remaining unbound to the particles in the supernatant
NA11l was quantified from the radiation dose of 32p, and DNAJ
As a result of calculating the amount of immobilized I, it was found that a lot of DNA strands were immobilized on 12 g of polyGMA particles.

Even if the resin on the surface of the carrier is a polymer made of the same 1,000 polymers, a carrier with a resin having an epoxy group on its surface can immobilize more DNAAl1i than a carrier with a resin without an epoxy group on its surface. I understand.

Actual Difference A Make 20 d of stock buffer in which the DNA-immobilized microspheres obtained in Example 3 are dispersed (the amount of DNA-immobilized microspheres is approximately 0.25 g), place in a 1.5-Eppendorf tube, and add 100 d of distilled water. Particle washing buffer (50 mM) Lis-HCl (pH 8,0)-baM ethylenediaminetetraacetic acid LIMDTT, 20% glycerol, 0.1% p-n-octylphenyl ether)
Washed 3 times with 100Δ.

HeLa lateral vacuoles were treated with 0.5mM MgCJb, ImMDTT.
After washing with PBS containing cell membrane disruption buffer (10
mNHEP E S (pH 7,9), 10mM
KCIIL 1.5ml MgO12,0
, 51DTT), homogenized, centrifuged to remove the supernatant, and further treated with nuclear protein extraction buffer (20dTT).
HE P E S (pH 7,9) - 25% glycerol.

0.42M NaCJL 1.5mM M+6:lb
, 0.2 Cod EDTA, 0.51 MPMSF, 0.5
■M DTT), centrifuged, and the supernatant dialyzed.
A nuclear grain extract with a protein concentration of 8 mg/di was obtained.

Poly [dl-dC 10d
and 10% p-n~octylphenyl ether 0.54
After stirring, the solution was left to stand for 15 minutes, added to the washed DNA-immobilized microspheres, and left to stand for 30 minutes. 4. TF
3 was bound to the DNA strands of the DNA-immobilized microspheres.

This mixture of nuclear particle extract and DNA-immobilized microspheres was centrifuged in a 1.5-Eppendorf tube, the supernatant was separated, and the pellet was washed three times with particle washing buffer. Thereafter, the pellet was mixed with elution buffer (50mM), salt II (pH 8,0)-1 cod ethylenediamine tetraacid,
E4TF3 was purified by washing three times with 204 (1.0M potassium chloride, 1mM DDT, 20% glycerol, 0.1% p-n-octylphenyl ether). 5
It was confirmed by DS-PAGE that six protein bands of E4TF3 were separated and IW-produced very selectively.

Comparison J2L3 Instead of the stock buffer 20d in which the DNA-immobilized microspheres obtained in Example 3 were dispersed (the amount of DNA-immobilized microspheres was approximately 0, 25+ig), D obtained in Comparative Example 2 was used.
NAIJ constant microspheres stock loose lI
20U of liquid! j (the amount of DNA immobilized microspheres is approximately 0,
E4T was prepared in the same manner as in Example 6 except that 25 mg) was used.
An attempt was made to purify F3. +E by 5DS-PAGE
Six protein bands of 4TF3 were confirmed, but
At least five other proteins were detected.

The DNA immobilized microspheres of the present invention are similar to conventional DNA
Compared to fixed microspheres, protein! It was found that the selectivity was better in No. 11.

Claims (1)

[Claims] 1. DN having a base sequence that specifically binds a specific protein to a carrier made of a resin whose surface has an epoxy group
A DNA immobilized microsphere in which the A chain opens and bonds the epoxy group, and does not adsorb proteins other than the base sequence part that specifically binds a specific protein in the DNA chain. 2. The DNA immobilized microsphere according to claim 1, wherein the specific protein is a transcriptional control factor. 3. The DNA immobilized microsphere according to claim 1 or 2, wherein the surface is made of polyglycidyl methacrylate. 4. DN having a base sequence that specifically binds a specific protein to a carrier made of a resin whose surface has an epoxy group
The A chain opens the epoxy group and bonds to the DNA immobilized microspheres, which do not adsorb proteins other than the base sequence part that specifically binds a specific protein in the DNA chain, through the DNA chain. A method for purifying a specific protein, which involves adsorbing the protein, removing other proteins, and then dissociating the protein from the DNA chain. 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 or 5, wherein the surface is made of polyglycidyl methacrylate.