JPH04210699A - Apparatus for separation of protein - Google Patents

Abstract

PURPOSE:To provide the subject apparatus equipped with a concentration-variable liquid supply unit, a protein separation unit containing an immobilized nucleic acid capable of adsorbing various kinds of proteins in a sample, a solution-transport unit for sending a solution thereto and a protein-receiving unit, and capable of precisely separating specified kinds of proteins in a short time. CONSTITUTION:A buffer solution is sent and transferred through a solution-transport pass 16 to a protein separation unit 8 by a concentration-variable liquid supply unit 1 capable of supplying a liquid while varying the concentration of the solution with time. A sample solution of the protein is injected through a sample inlet 9 attached to the solution-transport pass 16 and introduced into a protein separation unit 8 packed with an adsorbent prepared by fixing a nucleic acid capable of specifically adsorbing a specified protein to a rigid carrier having an enough strength to endure the pressure applied during supply or passage of the solution without deformation destruction. The specified protein is adsorbed thereby and an eluent is subsequently introduced to the protein separation unit 8 to elute the adsorbed protein. The eluted protein is then discharged through a solution outlet 11 to a protein-receiving unit 15, thus separating and recovering the objective protein.

Description

[Detailed description of the invention]

[00011

[Industrial Application Field] The present invention relates to a protein separation device used in the field of molecular biology, etc., for precisely and quickly separating proteins that specifically bind to nucleic acid sequences consisting of protein solutions. It is something. [0002] [Prior Art] As a conventional technique for separating DNA-binding proteins, open column chromatography (affinity chromatography, gel filtration) is used.
There is a method to separate substances by separating a protein solution by utilizing the difference in partition coefficient between a fixed protein layer and a moving protein layer, and dividing both parts of this solution into containers such as test tubes over time ( Prior art 1). In addition, a method has been adopted in which after gel electrophoresis is performed on a sample solution, the gel in a specific band is cut out and the substances contained in the sample solution are fractionated (Conventional technology 2).
. Furthermore, the sample solution is added to a centrifugal tube prepared with a density gradient using sucrose, etc., and centrifuged at 10,000 to 10,000 revolutions/minute to fractionate the substances in the sample solution according to their density. A method has also been adopted in which the fractionated substance is then taken out with a syringe needle and fractionated (Prior Art 3). [0003] [Problems to be Solved by the Invention] In Prior Art 1, an oven column using a so-called soft gel (for example, Sepharose: trade name manufactured by Pharmacia) made of a soft carrier such as a polysaccharide such as cellulose or agarose is used. I was using it. However, since soft gels generally have low physical strength, when high hydrostatic pressure is applied, the separation ability decreases due to structural changes in the gel, such as structural destruction. On the other hand, at low hydrostatic pressure, the speed of liquid delivery is slow, and as a result, substances once separated in the column are unable to maintain a precise concentration gradient due to the influence of diffusion, making it difficult to obtain high resolution. However, it also took a long time to separate. [0004] Furthermore, if solutions containing different types of proteins that flow out from a column sequentially are fractionated into a container with a fixed volume (for example, 5 ml), for example, when switching to the next container, a specific type of protein separated into the solution will be collected. When a large amount of protein is contained, that particular type of protein may be separated into another container or mixed with other types of protein separated into the next container. By further reducing the volume of the container for fractionation, confusion with other proteins can be prevented to some extent, but there is a limit to precise separation.
Moreover, the operation was extremely complicated. [0005] Conventional technology 2 allows proteins to be precisely separated, but it is very difficult to extract proteins from band portions. In Prior Art 3, the operation of extracting separated proteins with a syringe needle required extremely careful attention and was difficult. [0006] The present invention has been made to solve the problems in the prior art described above, and provides an apparatus for separating proteins precisely and in a short time. [0007]

[Means for Solving the Problems] The gist of the present invention is to provide a variable concentration liquid feeding unit that feeds a solution while changing its concentration over time, and a protein separation device that separates various proteins in a protein solution sample. a solution transport path for transporting the solution from the variable concentration liquid feeding section to the protein separation section; and a protein receiving section that receives over time the eluent containing the protein separated by the solution in the protein separation section. A protein separation device comprising: a sample injection port for injecting a protein solution sample into the protein separation section or the solution transport path; and a solution outflow port for flowing the eluent into the protein receiving section. In this, the protein separation section has an adsorbent formed by immobilizing a nucleic acid capable of specifically adsorbing a specific protein on a hard support having sufficient strength to prevent deformation and destruction due to the pressure during supply and passage of the solution. A protein separation device is characterized by the following. [00081 Hereinafter, the present invention will be explained with reference to the drawings. In the present invention, for example, a device configured as shown in FIG. 1 can be used as a variable concentration liquid feeding section that can feed a solution while changing its concentration, composition, ionic strength, pH, etc. over time. . That is, in FIG. 1, the variable concentration liquid feeding section 1 accommodates a solvent and a solute of a solution capable of dissociating the protein specifically adsorbed on the adsorbent 6 of the protein separation section 8 from the adsorbent 6. It is comprised of a container A2 and a container B3, a solution mixing section 4 and a pump 5 that mix the solvent and solute from the containers A2 and B3 while changing the mixing ratio. Container A2 and container B3 may each contain solutions with different concentrations. [00091] In the present invention, the protein separation section 8 includes, for example, a column 8 in which a chromatography tube 7 is filled with an adsorbent that adsorbs a specific protein. The adsorbent used in the protein separation section column 8 is, for example, an inorganic polymer (silica, porous glass, etc.), a natural or synthetic organic polymer (ebonite, melamine, polyetheretherketone, etc.), which can be immobilized with nucleic acids. The substance has a functional group, or a substance into which a functional group can be introduced by physical or chemical methods, or the surface treatment of metals (titanium oxide, etc.), ceramics (silicon nitride, etc.), crystal plates (salt, etc.) A nucleic acid that binds to a specific protein is immobilized on hard carrier particles that have been treated to allow nucleic acid to be immobilized, preferably at the ends thereof. The strength of the hard carrier is determined by the hydrostatic pressure during solution supply and passage1
It has enough strength to withstand 0 Kg/Cm2, preferably 100 Kg/Cm2. The diameter of the particles is preferably 10 ILm or less. The reason is,
This is because if the particle size is 10 μm or less, a sufficient surface area of the particles within the adsorbent can be obtained.

For example, when the restriction enzyme Eco RI contained in a protein solution sample is separated using the apparatus of the present invention, the adsorbent used is one in which DNA having the following sequence is bound and immobilized on particles. [00111 Note that the site to which the restriction enzyme Eco RI binds is underlined. [0012T The above DNA was added to silica particles (particle size: 5 μm)
For example, a method for immobilizing DNA on a DNA affinity column and preparing a DNA affinity column is performed as follows. After drying 100 μm each of purified DNA having the above sequences (a) and (b) under reduced pressure, it was dissolved in 100 μl of IMNaCl, and 10 μl of 0.4 M NaHCo3 (pH 7,5) was added.
Add 0 μm. Thereafter, it is treated on an aqueous solution at 95° C. for 2 minutes, and then annealed by cooling to 55° C. over 20 minutes. Add to this 20mg of Tresil activated silica gel.
and react at room temperature for 24 hours. After the reaction was completed, the gel was washed three times with IM NaCl, and the residue after centrifugation was collected as D
Immobilize NA. This was added to buffer A (0.5M NaC
1, pH 7, 5, 1mM EDTA, 10mM phosphoric acid), put it in a packer, and add buffer B (150
1mM NaCl, 10mM phosphoric acid) and fill it into the chromato tube 6. Buffer C (40mM Tris-
HCI, pH 7,5゜0.2M NaCl 1,5mM
EDTA, 2mM DTT) is sent to wash the column to obtain a DNA affinity column. The adsorbent 6 in the [00131 column is considered to be something like the partially enlarged view shown in FIG. 2. That is, the ends of the nucleic acids 22 are bonded and immobilized to the carrier 21, which is a support. Note that the substance that is immobilized on the carrier particles and that specifically binds to a specific protein is not limited to DNA synthesized using a synthesizer, and natural DNA, RNA, etc. can also be used. The chromatography tube 7 of the column weighs 100Kg.
It is not particularly limited as long as it can withstand the hydrostatic pressure of /Cm2 and is inert to the solution. For example, a metal tube (
Stainless steel, etc.), plastics (high-density polyethylene, etc.), and inorganic materials (glass, etc.) can be used. The internal cross-sectional area of the chromatography tube 7 is preferably 4 mm2 or less, since this allows for more precise protein separation. [0014] A possible reason for this is that 4 mm
This is believed to be because when the adsorbent is less than 2, the diffusion of protein in the radial direction is suppressed when the above-mentioned adsorbent is used in the present invention. In particular, when the inner cross-sectional area is 1 mm" or less, it is more preferable because it is affected by the viscosity of water and the hydrostatic pressure applied becomes high.
It can be injected into the column using, for example, the syringe 10. [00151 In the present invention, the protein receiving section 15 may be a device that sequentially spreads a solution containing separated proteins on the membrane 13 as shown in FIG. 1 using a rotor 14, for example. Alternatively, the direction of the eluent 12 made of the solution spread on the flat plate surface may be straight or spiral, and is not particularly limited. In the case of a device developed on the membrane 11, the material of the membrane 11 is, for example, a natural or synthetic organic polymer material such as nitrocellulose, nylon, polytetrafluoroethylene, or polyethylene, or an inorganic polymer material such as silicone resin. Molecules, metals such as aluminum can be used. [0016] [Example] Using a protein separation apparatus equipped with a variable concentration liquid feeding section 1 consisting of a container A2, a container B3, and a solution mixing section 4 shown in FIG. Buffer D (40mM Tris-HCI, 1.5
M Nacl, pH 7,5, 5mM EDTA, 2
mM DTT) with a concentration gradient (temporal concentration change) of 1
The buffer solution was mixed so that the concentration ranged from 0% to 100% in 0 minutes, and the solution was fed at a flow rate of 0.3 ml/min to the protein separation section 8 via the solution transport path 16. [00171 Protein separation unit 8 used in this example
is the inner diameter 2. Chromatography tube 7 with a diameter of 1 mm and a length of 100 mm
(made of stainless steel) is filled with a gel formed by fixing DNA that specifically binds to the restriction enzyme Eco RI to a silica gel having a particle size of 5 μm at its end. [0018] Before transporting the above solution, as a protein solution sample, Eco
Using a crude protein solution containing RI, use a syringe to inject 200 μm from the sample injection port provided at one end of the protein separation section.
Inject Eco into the gel and react at 15°C for 1 hour.
specifically binds RI. The above solution was passed through the protein separation section 8 for 10 minutes to elute Eco RI. Next, a solution containing Eco RI was allowed to flow out from the solution outlet 11 provided at the other end of the protein separation section 5 and developed in the protein receiving section 15 . [00191 The protein receiving section 15 provided in this example is composed of a nitrocellulose membrane (manufactured by Amajam) that moves at a speed of 1.5 cm/min as the membrane 13. The nitrocellulose membrane has a width of 50 mm. The length is 300 mm, and the binding solution (HEPES:
10mM, NaCl: 50mM, MgCl2:
10mM, EDTA: 0.1mM, DTT: 1mM
, skim milk powder (2.5%)). [00201 Confirmation that Eco RI was separated from this example was performed by the following method. A DNA duplex with a sequence that binds to Eco RI is synthesized and labeled with 32p. The nitrocellulose membrane obtained in this example was immersed in a 0.2 M KCl binding solution containing 105 cpm/ml of this labeled DNAAlX, and the DNA and protein were allowed to react.
．． After washing with 2M KCL binding solution for 1 hour, measurement was performed by autoradiography. As a result, the number of colored areas was 1
It was spot on. From this, according to this example, Ec
. It was confirmed that RI was specifically eluted and separated. [00211] [Effects of the Invention] According to the present invention, a specific type of protein can be separated precisely and in a short time, and moreover, the amount of mother liquor can be reduced compared to the conventional technique.

[Brief explanation of the drawing]

[Figure 1] Schematic diagram showing one embodiment of the present invention

2 is a partially enlarged view of an adsorbent used in the protein separation section of the example shown in FIG. 1. FIG.

[Explanation of symbols]

■...Variable concentration liquid feeding section 2...Container A 3...Container B 4...Solution mixing section 5...Pump 6...Adsorbent 7...Chromato tube 8...Protein separation 9...Sample injection port 10...Syringe 11...Solution outlet 12... Eluent 13... Menplan 14...Rotor 15... Protein receiving part 16... Solution transport path 21... Carrier 22...Nucleic acid

[Figure 1]

Claims (6)

[Claims] 1. A variable concentration liquid feeding section capable of feeding a solution while changing its concentration over time, a protein separation section separating various proteins in a protein solution sample, and a solution from the variable concentration liquid feeding section. a solution transport path that transports the protein to the protein separation section, and a protein receiving section that receives over time the eluent containing the protein separated by the solution in the protein separation section, the protein separation section or the solution In the protein separation apparatus, the transport path is provided with a sample injection port for injecting a protein solution sample, and a solution outlet is provided for flowing the eluent into the protein receiving section, wherein the protein separation section supplies the solution, A protein separation device comprising an adsorbent formed by immobilizing a nucleic acid capable of specifically adsorbing a specific protein on a hard carrier having sufficient strength to prevent deformation and destruction due to pressure during passage. 2. The protein separation apparatus according to claim 1, wherein the protein separation section is a column using a chromatography tube having an internal cross-sectional area of 4 mm^2 or less. 3. The protein separation apparatus according to claim 2, wherein the internal cross-sectional area of the chromatography tube is 1 mm^2 or less. 4. The protein separation device according to claim 1, wherein the nucleic acid capable of specifically adsorbing a specific protein is immobilized on a hard carrier at its terminal end. 5. The protein separation device according to claim 1 or 4, wherein the hard carrier has a strength capable of withstanding hydrostatic pressure of 10 kg/cm^2. Claim 6: The hard carrier has a hydrostatic pressure of 100Kg/Cm^2
The protein separation device according to claim 5, which has strength enough to withstand