JP2006342154A - Protein fractionation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To fractionate a number of low-molecular-weight proteins from a solution containing two or more kinds of proteins, such as blood serum and plasma, and to recover specific proteins among the low-molecular-weight proteins. <P>SOLUTION: A protein fractionation device is equipped with a purification module for purifying proteins in a solution treated by a fractionation module filled with a hollow fiber, wherein the purification module is capable of selectively adsorbing and recovering specific proteins from the solution. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a protein fractionation device including a purification module that can selectively adsorb proteins from fractionated proteins. The present invention also relates to a method for fractionating proteins contained in a solution using the protein fractionation device.

  In recent years, proteomic analysis (proteomics) has attracted attention as post-genomic research. Since the protein that is a gene product is thought to be directly linked to the pathology of the disease, the research results of proteome analysis that comprehensively examines the protein are expected to be widely applicable to diagnosis and treatment. Moreover, it is highly probable that proteome analysis can find many pathogenic proteins and disease-related factors that could not be found by genome analysis.

  While conventional proteome analysis research has only comprehensively detected and identified proteins, more recently, research using a wide range of analysis methods such as analyzing post-translational modifications of these proteins has been conducted. It has begun to attract attention. It is known that proteins are subjected to various modifications such as phosphorylation, glycosylation, acetylation, lipid addition, and ubiquitin addition after being synthesized in cells. The modification greatly affects the activity of the protein, and thus affects the behavior of the entire protein group. When a disease develops in vivo, the expression level of multiple proteins themselves changes, and the post-translational modification state of some proteins changes, thereby causing normal behavior of the proteins. Is believed to be lost. For example, protein phosphorylation is deeply involved in intracellular signal transduction in biological phenomena and is thought to be deeply involved in the development of diseases such as canceration of cells. Post-translational modifications of these proteins can never be analyzed from genomic information, and are important analysis targets for proteome analysis. For a comprehensive analysis of post-translational modifications of proteins, first identify the modified protein itself, determine which part of the protein is modified, and investigate how much modification has been applied. Analysis is the main flow.

  The rapid progress of such proteomic analysis is technically because high-speed structural analysis using a mass spectrometer (MS) has become possible. Practical use of MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) enables high-throughput ultra-trace analysis of polypeptides, and enables trace amounts of proteins that could not be detected to be detected in a short time. It has become possible to identify a very wide variety of measurements. In addition, for analysis of post-translational modifications, it is becoming possible to detect modified proteins and identify their modified sites by setting appropriate measurement conditions. These analytical techniques are now very powerful tools in the search for disease-related factors.

  The primary purpose of applying proteome analysis to clinical practice is to discover biomarker proteins whose expression level and modification state vary greatly depending on the disease. Since the biomarker behaves in relation to a disease state, it can be a diagnostic marker and is also highly likely to be a drug discovery target. In other words, since the results of proteome analysis are directly linked to the creation of diagnostic markers and drug targets, it will become a diagnostic / treatment trump (evidence) technology in the post-genomic era. Furthermore, since the identified biomarkers lead directly to benefits that patients can enjoy, such as drug responsiveness assessment and prediction of side effects, they will play a major role in promoting so-called tailor-made medicine. I can say that.

  In proteome analysis (clinical proteomics) in clinical research, it is required to analyze a large number of specimens quickly and reliably. Moreover, since clinical specimens are very small and valuable, it is necessary to perform high-resolution, high-sensitivity, and high-function measurements quickly. Mass spectrometry has become a major driving force, and this greatly contributes to the ultra-high sensitivity and high throughput characteristics of the mass spectrometer as described above. However, although the methods and instruments have been rapidly improved, the current situation is that the proteome analysis is not easily and quickly performed at the clinical site.

  One of the causes is the need for pretreatment of clinical specimens. Prior to analysis by mass spectrometry, it is necessary to fractionate and purify the protein of a clinical specimen, but this treatment actually takes several days, and the pretreatment operation is complicated and requires experience. It has become a major obstacle to clinical application. Although it is extremely useful if a systemic disease can be diagnosed and pathologically managed from a small amount of blood or body fluid, it has many problems due to the diversity of proteins contained in serum and plasma. Is the current situation.

  Although it is estimated that there are more than 100,000 human proteins, it is said that the number of proteins contained in serum alone is about 10,000, and the combined serum concentration is estimated to be about 60-100 mg / mL. Is done. High-content proteins in serum are albumin (molecular weight 66 kDa), immunoglobulin (150-190 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several hundred kDa), etc., all of which are in large quantities (> mg / mL). On the other hand, most of physiologically active proteins such as peptide hormones, interleukins, cytokines and the like, which are considered to be pathological biomarkers and pathogenesis-related factors, are present in very small amounts (<ng / mL). The content ratio is indeed in the nano-pico level compared to the high content component of high molecular weight. From the viewpoint of protein size, 70% or less of all types of proteins have a molecular weight of 60 kDa or less, and the above-mentioned extremely small amount of biomarker protein is almost always included in this region (for example, Non-Patent Document 1). . Since these proteins pass through the kidney and are partially excreted in urine, it is possible to measure not only blood but also urine as a specimen.

  Regarding post-translational modification, depending on the type of modification, the proportion of proteins that are actually modified in cells is very small. For example, in the case of protein phosphorylation, the proportion of proteins that are actually phosphorylated is very small compared to the total amount of protein. Therefore, even when trying to analyze the phosphorylation modification of proteins obtained from biological samples such as serum and plasma, it is buried in the signal of high-content, high-molecular-weight unmodified proteins such as albumin and IgG, and only modified proteins are accurately detected. Actually, it is difficult to measure.

  Therefore, in order to perform proteome analysis in a general serological test, it is essential to exclude high-content, high-molecular-weight protein components such as albumin and IgG that interfere with pathogenesis-related trace component detection. Currently, high-performance liquid chromatography (LC) and two-dimensional electrophoresis (2D PAGE) are used as means for separating these proteins. It takes ~ 2 days. This required time is much longer than the analysis time of several minutes such as MALDI-TOF-MS and ESI-MS (electrospray ionization mass spectrometry), and the great advantage of high throughput of MS can be fully demonstrated in clinical proteome analysis. I'm not there. For this reason, for the purpose of obtaining analysis results in the shortest possible time for diagnosis and treatment in the medical field, it must be said that it is extremely impractical at the present time, and it is difficult to use MS for daily clinical tests. It has become a big cause.

  If this point is solved, it can be expected that the speed of diagnosis of clinical tests by clinical proteome analysis will be dramatically improved. Specifically, a device capable of fractionating and separating a target protein group at a high speed from a very small amount of sample may be used as an alternative to LC or 2D-PAGE.

  Products that have already been put to practical use as a substance to be removed from albumin or disclosed techniques include a carrier on which an affinity ligand such as a blue dye is immobilized (for example, Nihon Millipore: “Montage Albumin Deplete Kit (registered trademark)) "Nippon Bio-Rad: Affi-Gel Blue (registered trademark) gel), a centrifugal tube type filtration concentration unit that fractionates high molecular weight components by centrifugal filtration (for example, Nihon Millipore:" Amicon Ultra (registered trademark) ) ", Sartorius:" Vivapin "), methods of fractionation by electrophoretic principles (e.g., Gradipour:" Gradiflow® "system), traditional precipitation methods such as ethanol precipitation of Cohn and chromatography. Fractionate Law (for example, Non-Patent Document 2), and the like. In addition, there is a report (for example, Non-Patent Document 3) in which albumin and IgG in plasma are removed using a carrier on which an anti-albumin antibody is immobilized and a carrier on which protein G is immobilized, and analysis is performed by two-dimensional electrophoresis. In addition, a product that simultaneously removes albumin and immunoglobulin G (IgG) has been marketed (GE Healthcare: Albumin and IgG Removal kit). In addition, there is a report of analyzing the albumin removal efficiency of these representative albumin removal products by comparing the removal efficiency of albumin by two-dimensional electrophoresis (Non-Patent Document 4). However, these are all insufficient for separation and fractionation, unsuitable for trace samples, diluted samples, elution of immobilized antibodies, mass spectrometry, etc. The actual situation is that there are problems such as the mixing of drugs that become obstacles.

  Examples of products for separating proteins by electrophoresis include “Model 491 Prep Cell” and “Mini Prep Cell” from Biorad, “Prep Foresis (registered trademark)” from Ato Corporation. These products are separated by separating the protein applied to the top of the cylindrical gel by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and collecting the separated protein from the bottom of the gel. . Although these products have the advantage of being able to process a certain amount of protein at a time, it takes time and effort to prepare an electrophoresis gel, and the unreacted sample contained in the gel in the collected sample. Since impurities such as acrylamide may be mixed, it is not suitable as a sample for mass spectrometry.

  Techniques for concentrating protein samples include a method using an ultrafiltration membrane, a method of precipitating and re-dissolving in a small amount of solvent, a method of freeze-drying and re-dissolving in a small amount of solvent, and absorbing the solvent with a dry gel There are methods. The method using an ultrafiltration membrane is a commonly used technique. Specifically, a centrifugal tube type filtration concentration unit (for example, Nihon Millipore Corporation) for concentration by filtering a protein solution sample by centrifugation. : “Amicon Ultra (registered trademark)”, Sartorius: “Vivapin”. However, there is a distribution in the pore size of the membrane, and there are pores through which a substance with a molecular weight higher than the specified molecular weight can pass. Therefore, in fact, proteins having a molecular weight higher than the molecular weight cutoff of the membrane pass through the pore. In reality, it may leak into the filtrate, and thus a reduction in recovery rate is unavoidable.

In addition, as a technology for fractionating and purifying a modified protein, a product or technology that has already been put into practical use includes a method using beads immobilized with an antibody that specifically binds to a chemical modification group for analysis. In general, a method using beads immobilized with a substance that specifically binds to each chemical modification, such as a metal complex or a lectin, is generally used. Commercially available or disclosed commercially available products include PhosphoProtein Purification Kit (manufactured by Qiagen), Proteo Extract Phosphopeptide Capture Kit (manufactured by Calbiochem Corp.), and PhosphoprotemTenproKimenQ. Kit (manufactured by Qiagen) and the like. However, in these methods, high-content and high-molecular weight proteins such as the above-mentioned albumin non-specifically adsorb to these beads, which inhibits binding of modified proteins for analysis to the beads. Moreover, there is a problem that it is difficult to specifically recover only the phosphorylated protein because these high-content proteins are eluted together during the recovery of the modified protein. Furthermore, the method using the beads on which the antibody is immobilized has a problem that it is difficult to produce an antibody having strictly high binding specificity for the chemical modification. For these reasons, it is very difficult to specifically recover only the modified protein from the biological sample.
By Anderson NL, Anderson NG, "The Human Plasma, Prospectome: History, Character, and Prospectome: history, character ”, Molecular & Cellular Proteomics, 2002, Vol. 1, p 845-867. Edited by the Japanese Biochemical Society, “Seminar in Experimental Chemistry of Voluntary Chemistry (Vol. 1) Protein (1) Separation, Purification, and Properties”, Tokyo Kagaku Doujin, 1990 Carrie Greenough et al., “A method for the rapid depletion of albumin and immunity of immunoglobulin.” human plasma) ", Proteomics, 2004, Vol. 4, p3107-3111. Chromy BA et al., “Proteomic Analysis of Human Sheram by Two-Dimensional Differential Gel Electrophoresis After Depletion of High-Abundant Proteins ( Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant protein (Journal of Proteor).

  In view of the problems of the prior art, the present inventors have intensively studied. As a result, when fractionating at least two kinds of proteins contained in a solution, the present invention is classified by a fractionation module filled with a hollow fiber membrane. From the fractionated proteins, the inventors have found that specific proteins can be recovered by adsorbing them to one or a plurality of linked purification modules and eluting them using an elution buffer, thereby achieving the present invention.

  That is, an object of the present invention is to provide a protein fractionation device including a purification module that can selectively purify a protein from fractionated proteins by an adsorption method. Another object of the present invention is to provide a protein fractionation method using the protein fractionation device.

In order to achieve the above object, the present invention has the following configuration.
“(1) A protein fractionation device comprising a purification module for recovering a protein in a solution treated by a fractionation module filled with a hollow fiber membrane, wherein the purification module is a specific protein in the solution. A protein fractionation device characterized by having a function of selectively adsorbing and recovering the protein. "
“(2) The protein fractionation device according to (1), wherein a solution containing the protein treated by the fractionation module is circulated to the purification module for treatment.”
“(3) The protein fractionation device according to (1) or (2), wherein the fractionation module and the purification module are connected by a solution channel and can be operated continuously.”
“(4) The purification module is composed of a metal chelate resin, a protein immobilization resin, an antibody immobilization resin, a lectin immobilization resin, a group-specific affinity resin, and a hydrophobic compound immobilization resin, an ion exchange resin, and a reverse phase resin. The protein fractionation device according to any one of (1) to (3), wherein the protein fractionation device comprises one or more resins selected from the group consisting of:
"(5) The protein fractionation device according to any one of (1) to (4), comprising a plurality of purification modules containing different resins."
“(6) The protein fractionation device according to any one of (1) to (5), wherein all the purification modules are connected to the solution flow path.”
“(7) The protein fractionation device according to any one of (1) to (6), wherein a specific protein is collected as a fraction.”
“(8) The protein fractionation device according to any one of (1) to (7), wherein 20% or more of a specific protein is recovered.”
“(9) The protein fractionation device according to any one of (1) to (8), wherein the solution containing the protein is a body fluid containing the protein.”
“(10) The protein fractionation device according to any one of (1) to (9), wherein the specific protein is a disease marker protein.”
“(11) A method for fractionating a protein-containing solution using a protein fractionation device according to (1) to (10) according to the difference in molecular weight.”

  According to the present invention, at least two kinds of proteins contained in a solution such as serum and plasma can be fractionated by the difference in their physical properties, and a specific protein is further extracted from the fractionated proteins. A specific protein can be recovered by adsorbing to the purification module and eluting the protein adsorbed to the purification module using an elution buffer.

  The present invention relates to a protein fractionation device comprising a purification module for recovering a protein in a solution treated by a fractionation module filled with a hollow fiber membrane, the purification module comprising a specific protein in the solution. It is a protein fractionation device characterized by having a function of selectively adsorbing and recovering.

  “Fractionation” as used in the present invention refers to separation of solutes in a solution, and refers to separation of all or part of solutes when a plurality of types of solutes are contained. For example, when preparing a sample for analyzing a body fluid component, a recovered component and a discarded component are discriminated. In addition, the “fractionation module” in the present invention is an instrument used for the purpose of discriminating proteins in a solution based on differences in physical properties. That is, by using the fractionation module of the present invention, proteins can be fractionated into two or more groups based on differences in physical properties such as molecular weight and isoelectric point.

  In the present invention, the hollow fiber membrane filled in the fractionation module has pores in which a low molecular weight substance contained in the liquid can move bidirectionally with the membrane as an interface. The method for producing a hollow fiber membrane having such a function is not particularly limited as long as it is a commonly used method for producing a hollow fiber membrane such as wet spinning and dry / wet spinning. For example, when forming a hollow fiber membrane using a double ring die, a polymer fluid is spun into a hollow fiber shape using a die, a porous material is formed in the flow-down process, and solidified by passing through a coagulation bath. When the core side fluid is washed with a washing bath, dried and crimped, and wound up, a hollow fiber membrane is obtained. These operations are preferably performed online. In addition, it is preferable that the obtained hollow fiber membrane does not have surface scratches, collapse of the hollow portion due to bending, pressing, or the like.

  The hollow fiber membrane used in the present invention can be used for both a dry type and a wet type. When using a dry type hollow fiber membrane, it is preferable to prevent drying by treating the surface of the membrane with glycerin or the like.

  In the present invention, the material of the hollow fiber membrane filled in the fractionation module is not particularly limited as long as it is a material that can industrially form a hollow fiber membrane. For example, polysulfone (PS), polyethersulfone (PES), polymethacryl Methyl acid (PMMA), polyacrylonitrile (PAN), polycarbonate (PC), polyamide, aromatic polyamide, polytetrafluoroethylene (PTFE), polyvinylidene fluoride, polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP ), Synthetic polymers such as polyimide, polyvinyl alcohol (PVA), ethylene-vinyl alcohol copolymer and their derivatives, copolymers or composites containing them, or cellulose, cellulose acetate, cellulose triacetate, cellulose nitrate, Diethylamino Natural polymers and their derivatives such as chill cellulose, copolymers or complexes containing them is preferably used.

  Also, two or more of these materials may be mixed, and various additives may be mixed for the purpose of improving the properties of the materials. For example, it is preferable that a hydrophilic polymer or a hydrophobic polymer is blended with a polymer that is a material for the hollow fiber membrane to form a membrane, thereby making the surface of the hollow fiber membrane hydrophilic or hydrophobic.

  Also preferred are a method of coating the surface of the hollow fiber membrane with a hydrophilic polymer or a hydrophobic polymer, and a method of graft polymerization. Examples of the hydrophilic polymer used here include polyethylene glycol, polyvinyl pyrrolidone, polyethylene imine, polyvinyl alcohol, polyacrylamide, poly (hydroxyethyl acrylate), polyvinyl methyl ether, polyacrylic acid, polyvinyl pyridine, poly (N-vinylacetamide). ), 2-methacryloyloxyethyl phosphorylcholine polymers and synthetic compounds such as metal salts and esters thereof, starch, hydroxyethyl cellulose, carboxymethyl cellulose, alginic acid, hyaluronic acid, polyglutamic acid, chitosan, lignin, polylysine, silk fibroin, carrageenan, Natural compounds such as casein, glycolic acid, collagen, gelatin and their metal salts and esters are preferably used. In addition, you may combine the above-mentioned synthetic polymer compound and a natural polymer compound. As other hydrophilization methods, an oxygen plasma irradiation method or a polysilazane coating method can be used. On the other hand, as a hydrophobizing method, silicon coating, fluorine coating, and surface treatment by plasma irradiation are preferably used. Protein adsorption can be suppressed by either the hydrophilization or hydrophobization method described above.

  The “specific protein” in the present invention refers to a protein to be analyzed among the proteins fractionated by the fractionation module. For the purpose of analyzing proteins that have undergone post-translational modifications, for example, for the purpose of analyzing proteins that phosphorylated proteins, glycoproteins, etc. bind to nucleic acids or low molecular weight compounds. If there is a nucleic acid binding protein, adenosine triphosphate (ATP) binding protein, etc., for the purpose of analyzing all the proteins fractionated by the fractionation module, all the proteins fractionated by the fractionation module are listed. However, it is not particularly limited to these.

  The term “selectively adsorbed” as used in the present invention refers to adsorbing a specific protein among the proteins fractionated by the fractionation module to the purification module according to the properties of the protein. Selective adsorption includes adsorption of phosphorylated protein to metal chelate group, adsorption of antigenic protein to antibody, adsorption of glycoprotein to lectin protein, adsorption of nucleic acid binding protein to nucleic acid, ion exchange group of protein However, the present invention is not particularly limited thereto.

  The term “purification” as used in the present invention refers to recovering a group containing a specific protein to be analyzed from proteins fractionated by a fractionation module. For example, when preparing a sample for analyzing blood components, it is possible to recover proteins such as phosphorylated protein, glycoprotein, and nucleic acid binding protein, but it is not particularly limited to these.

  The “purification module” as used in the present invention refers to selective adsorption of a specific protein from the protein fractionated by the fractionation module, and the adsorbed specific protein is eluted and recovered with an elution buffer. It refers to the instrument used for the purpose.

  The packing of the purification module in the present invention is not particularly limited as long as there is no eluate mixed into the protein sample and adversely affecting the measurement. Examples of preferred forms include beads, gels, fabrics, knitted fabrics, hollow fiber membranes, and flat membranes. When processing efficiency is taken into consideration, it is preferable to use beads and gels, and it is more preferable to use porous beads and gels that can prevent the pressure rise that occurs during operation as much as possible.

  The material of the filler of the purification module in the present invention is not particularly limited as long as it can form the beads, gel, fabric, knitted fabric, hollow fiber membrane, flat membrane and the like. The surface of the packing is preferably subjected to modification for adsorbing the protein. In this case, it is preferable that the protein is not adsorbed nonspecifically on the base material of the packing in order to exert the effect of the modification. . Therefore, in order to prevent non-specific adsorption of proteins, it is preferable to hydrophilize or hydrophobize the substrate surface of the packing.

  The shape of the purification module in the present invention is not particularly limited as long as the solution can pass through without leaking, and a packed column, an open column, a capillary column, and the like can be preferably used.

“Recovery” as used in the present invention refers to obtaining a specific protein eluted from the purification module in a solution in a container.
Furthermore, in the present invention, preferably, the composition of the elution buffer is changed and introduced into the purification module, the time during which each protein is eluted is changed, and the recovery container is changed according to the time and recovered. To “recover” as specific fractions of specific proteins. In order to change the composition of the elution buffer and introduce it into the purification module, mix two elution buffers with different compositions using a commercially available gradient forming device or a pump capable of gradient formation. Although the method of introducing while changing the ratio is preferably used, it is not particularly limited thereto.

  Although the apparatus used for collection | recovery of specific protein by this invention will not be specifically limited if a collection container can be changed for every time, Preferably a commercially available fraction collector is used.

  The material of the container for recovering a specific protein in the present invention is not particularly limited as long as it can recover the solution. For example, polysulfone (PS), polycarbonate (PC), polyamide, aromatic polyamide, polyvinyl chloride (PVC) Synthetic polymers such as polyethylene (PE) and polypropylene (PP) and derivatives thereof, and copolymers or composites containing them are preferably used. In order to suppress protein adsorption, it is also preferable to subject the inner surface of the container to hydrophilization or hydrophobization.

  The protein fractionation device of the present invention is preferably treated by circulating a solution containing the protein treated by the fractionation module to the purification module. By circulating the solution containing the fractionated protein to the purification module, the protein can be reliably adsorbed to the purification module.

  In the present invention, it is preferable that the fractionation module and the purification module are connected by a solution channel.

  The term “solution channel” as used herein refers to a pipe installed between the outlet of the fraction processed by the fraction module and the inlet of the purification module, and the fraction module and the purification module in the solution channel. This means that the fraction solution can be directly processed by the purification module. By doing so, the surface area in the circuit can be reduced, and loss due to adsorption of proteins to the circuit can be reduced. In addition, since the solution containing the fractionated protein can be directly processed, the operation is remarkably simplified.

  In the protein fractionation device of the present invention, the purification module preferably includes a resin on which a substance having a property of selectively adsorbing a specific protein is immobilized. By using a resin on which a substance having a property of selectively adsorbing a specific protein is immobilized, it is possible to efficiently adsorb the protein fractionated by selective interaction.

  In the present invention, the purification module is one selected from the group consisting of a metal chelate resin, a protein immobilization resin, a lectin immobilization resin, a group-specific affinity resin, and a hydrophobic compound immobilization resin, an ion exchange resin, and a reverse phase resin. It is preferable to include a resin selected from one or more types.

  In the present invention, the “metal ion chelate resin” means that a metal atom is coordinated to a polymer having a three-dimensional network structure in order to selectively recover a phosphorylated protein from a fractionated protein. Refers to a resin to which a chelate is immobilized. As the chelate group, a nitriloacetic acid group, an iminodiacetic acid group, or the like is preferably used. Moreover, the metal atom to coordinate is not specifically limited if it is a metal ion, For example, iron, zinc, gallium, zirconium, etc. are each used suitably. Moreover, it is preferable to try to adsorb and fractionate the fractionated phosphorylated protein more efficiently by preparing a chelate resin in which a plurality of types of metal atoms are mixed and coordinated.

  “Protein immobilization resin” as used in the present invention refers to a polymer having a three-dimensional network structure in order to selectively recover proteins that interact with other proteins from fractionated proteins. It is a resin with a specific protein immobilized on it. The protein to be immobilized is not particularly limited. For example, by using a resin with immobilized β2 microglobulin as a purification module, it covers proteins that interact with β2 microglobulin from low molecular weight proteins fractionated from serum or cell extracts using a fractionation module. Can be recovered automatically. In addition, it is preferable to immobilize a protein having enzyme activity so that the protein fractionated by the fractionation module can be adsorbed and the enzyme reaction can proceed simultaneously. Examples of the protein having enzyme activity include protease, nucleolytic enzyme, phosphorylase, dephosphorylase, glycosylation enzyme, sugar chain cleaving enzyme, restriction enzyme, etc. It is not limited to.

  In the present invention, “antibody-immobilized resin” refers to a polymer having a three-dimensional network structure in order to selectively recover antigen proteins that interact with specific antibodies from fractionated proteins. It is a resin on which a specific antibody is immobilized. The antibody to be immobilized is not particularly limited. For example, by using a resin with an anti-albumin antibody immobilized in the purification module, it is possible to comprehensively recover the remaining albumin from low molecular weight proteins fractionated from serum or cell extracts using a fractionation module. Can do. Further, by appropriately selecting the liquid to be filled in the module, the protein that interacts with the antigen protein can be similarly recovered. For example, in the case of a resin on which an anti-albumin antibody is immobilized, a protein that interacts with albumin is recovered together with albumin. Furthermore, depending on the type of protein, the antigen protein and the protein that interacts with the antigen protein can be collected separately as fractions by appropriately selecting the composition of the elution start buffer and the elution buffer. .

  The “lectin immobilization resin” as used in the present invention is an affinity for a sugar called a lectin in a polymer having a three-dimensional network structure in order to selectively recover a glycoprotein from a fractionated protein. It is a protein that has been immobilized. Examples of the lectin protein include red bean lectin (concanavalin), snowdrop lectin, inrenji lectin, lentil lectin, peanut lectin, elderberry lectin, wheat germ lectin, and the like. Absent. It is preferable to recover the glycoprotein more reliably by properly using these proteins, or by using a combination of resins on which a plurality of types of lectin proteins are immobilized.

  The “group-specific affinity resin” referred to in the present invention is a resin in which a low molecular compound that specifically binds to a part of a protein group is immobilized on a polymer having a three-dimensional network structure. Examples of the compound include benzamidine, adenosine diphosphate (ADP), adenosine triphosphate (ATP), oligonucleotide, glutathione, heparin, streptavidin, nucleic acid, blue dye, and the like, but are not particularly limited thereto.

  The “hydrophobic compound-immobilized resin” as used in the present invention means that a hydrophobic compound is immobilized on a polymer having a three-dimensional network structure in order to recover the hydrophobic protein from the fractionated protein. It is the resin that was made. Examples of the hydrophobic compound include, but are not limited to, a butyl group, a methyl group, a benzyl group, a phenyl group, an octyl group, an isopropyl group, and an ether group. Preferably, various proteins can be recovered by properly using these resins.

  The “ion exchange resin” referred to in the present invention is a resin in which a polymer having a three-dimensional network structure is provided with an ion exchange group for performing ion exchange. There are two types of ion exchange groups: cation exchange groups and anion exchange groups. Examples of cation exchange groups include carboxymethyl groups and sulfopropyl groups. Examples of anion exchange groups include: A diethylaminoethyl (DEAE) group, a diethylaminopropyl group, a quaternary ammonium group, a trimethylaminoethyl group, a trimethylaminopropyl group, and the like are each preferably used. By using different ion exchange resins depending on the composition and pH of the buffer solution, the fractionated protein can be adsorbed efficiently, and as a result, the fractionated protein can be recovered with high yield.

  The “reverse phase resin” referred to in the present invention is obtained by immobilizing a hydrophobic functional group on a polymer having a three-dimensional network structure in order to comprehensively collect fractionated proteins. As the functional group to be immobilized, a phenyl group, a cyanopropyl group, or a linear alkyl group is preferably used. A linear alkyl group having up to C2-C20 is used, and is generally commercially available, and particularly preferably C3 (trimethyl group), C4 (butyl group), C8 (octyl group), C18 (octadecyl group). ) Is used, and the fractionated protein can be recovered in high yield by properly using these functional groups.

The purification module in the present invention is effective even when used in a single stage, but it is preferable to include a plurality of purification modules containing different resins. By providing a plurality of purification modules, it is possible not only to reliably adsorb the proteins fractionated by the fractionation module, but also to connect and use purification modules loaded with different adsorbents to be adsorbed. Thus, it becomes possible to separate and purify according to the difference in protein properties.
When connecting many types of purification modules in parallel, it is preferable to connect them using a commercially available multi-position valve (for example, a multi-position valve manufactured by Valco) because three or more types of purification modules can be easily connected. In addition, if a bypass channel that is not connected to the purification module is provided, after the fractionation process is performed using only the fractionation module, the channel is switched to the purification module to connect the fractionation module and the purification module. To start adsorption of proteins to the purification module. In addition, it is preferable to circulate the protein solution that has passed through the purification module and pass it through the purification module, so that more protein can be reliably recovered.

  In the fractionation device according to the present invention, the purification modules are preferably connected to the solution flow path for processing.

  In the purification module of the present invention, it is preferable that all the purification modules are connected to the solution channel.

  By connecting the purification modules to the solution flow path, the solution containing the fractionated protein can be efficiently adsorbed to the purification module. Moreover, the surface area in a circuit can be reduced and the loss by adsorption | suction to the circuit of protein can be reduced. In addition, since the solution containing the fractionated protein can be directly processed, the operation is remarkably simplified.

  When the protein is fractionated using the protein fractionation device in the present invention, the following method is preferably used.

  First, a solution containing a protein charged into a circuit is fractionated by a fractionation module, and the fractionated solution is passed through a purification module to adsorb a specific protein according to the properties of the protein. At this time, in order to reliably adsorb a specific protein, it is desirable to install a plurality of purification modules.

  The liquid filled in the module and circuit in the present invention may be any as long as it can dissolve the protein. However, it is preferable that a substance that prevents the specific protein from adsorbing to the purification module is not included. For example, if the metal chelate resin is used for the packing of the purification module and the purpose is to selectively adsorb phosphorylated protein and then recover it, the phosphoric acid concentration of the filling liquid is preferably 0 to 5 mM, preferably 0 mM. More preferably. When the phosphoric acid concentration exceeds 5 mM, the phosphoric acid molecule inhibits the adsorption of the phosphorylated protein to the resin, which is not preferable. In addition, for example, for the purpose of exhaustively adsorbing and recovering proteins by using an ion exchange resin for the packing of the purification module, the salt concentration is preferably 5 mM to 1 M, and more preferably 10 to 30 mM. If the concentration is less than 5 mM, the buffering capacity of the buffer solution is impaired, and not only the pH changes easily but also the protein may precipitate. This is not preferable because it causes precipitation and clogging of the flow path.

  The liquid filled in the module and the circuit may be arbitrary as long as the above points are satisfied, and examples thereof include a buffer solution, water, a water-soluble organic solvent, and a mixture thereof. Examples of buffers that can be preferably used here include phosphate buffer, carbonate buffer, bicarbonate buffer, acetate buffer, citrate buffer, formate buffer, and borate buffer. Liquid, Tris-HCl buffer, and the like. Examples of the water-soluble organic solvent suitably used in the present invention include nitrogen-containing compounds such as acetonitrile and pyridine, cyclic ether compounds such as 1,4-dioxane and propylene oxide, ketone compounds such as acetone and ethyl methyl ketone, N , N-dimethylformamide, N, N-dimethylacetamide, N, N′-dimethyl-2-imidazolidinone, amides such as N-methyl-2-pyrrolidone, sulfur-containing compounds such as sulfolane and dimethyl sulfoxide, methanol, Monohydric alcohols such as ethanol, 2-propanol, cellosolves such as 2-methoxyethanol (methyl cellosolve), 2-ethoxyethanol (ethyl cellosolve), 2-aminoethanol (monoethanolamine), diethanolamine, triethanolamine, etc. D Noruamin, ethylene glycol, propylene glycol, diethylene glycol, and polyhydric alcohols such as glycerin. Furthermore, additives may be included for the purpose of improving the membrane permeability of proteins or increasing the permeability of low molecular weight proteins. Examples of additives that are preferably used in the present invention include protein denaturants, chaotropic ions, and the like. For example, urea, guanidine hydrochloride, and potassium thiocyanate are preferably used.

  The pH of the liquid filled in the module is preferably 4 to 10 so that the protein is not denatured and can exist stably, and more preferably 5 to 9 neutral. A pH lower than 4 or higher than 10 is not preferable because the protein may be denatured and precipitated. Furthermore, a pH that does not prevent specific proteins from adsorbing to the purification module is more desirable. For example, if the purpose is to specifically adsorb and purify phosphorylated protein using metal ions in the purification module, the pH of the fractionation buffer is preferably 5-8. A pH exceeding 8 is not preferable because the effect of adsorbing the phosphorylated protein on the purification module is weakened.

  The “elution buffer” as used in the present invention is not particularly limited as long as it is a solution that can dissociate the interaction of proteins adsorbed on the packing of the purification module. Further, the “elution start buffer” is not particularly limited as long as it does not interfere with the interaction of the protein adsorbed on the packing of the purification module. Additives such as salts, organic solvents, and low molecular weight compounds are added to the buffer solution for elution. In addition, the elution start buffer does not contain these additives, or it does not interfere with the interaction between the protein and the protein adsorbed on the packing of the purification module. It is also preferable to use the liquid filled in the module and the circuit as it is as a buffer solution for starting elution.

  For example, when a metal chelate resin is used as a packing for a purification module for the purpose of recovering phosphorylated protein, phosphate is added to the buffer solution. When adding phosphate, the phosphate concentration of the elution buffer is preferably 10 mM to 5 M, more preferably 50 mM to 1 M. The phosphate concentration of the elution start buffer is preferably 0 mM to 1 mM, more preferably 0 mM. In addition, for example, when a lectin immobilization resin is used as a packing for the purification module for the purpose of recovering glycoprotein, sugar is added to the elution buffer. In this case, the sugar concentration of the elution buffer is preferably 10 mM to 1M, more preferably 100 mM to 200 mM. In addition, the sugar concentration of the elution start buffer is preferably 0 mM to 5 mM. Further, for example, when an ion exchange resin is used, a salt is added to the buffer solution. In this case, the salt concentration of the elution buffer is preferably 10 mM to 10M, and more preferably 50 mM to 5M. The salt concentration of the elution start buffer is preferably 0 mM to 5 mM.

  In order to elute the adsorbed protein, prepare the elution buffer and elution start buffer by changing the pH of the buffer, and gradually weaken the interaction between the purification module packing and the protein. You can also. In this case, the concentration of a substance such as a salt, an organic solvent, or a low molecular compound added to the elution buffer can be lowered as compared with the case where the pH is not changed. For example, when an ion exchange resin is used, it is possible to elute proteins by adjusting the salt concentration of the elution buffer to a low concentration of 50 to 100 mM.

  As described above, a specific protein can be selectively recovered by treating a solution containing the protein using the “protein fractionation device” in the present invention. At this time, the recovery rate of the target specific protein is preferably 20% or more, and more preferably 30% or more. Further, when fractionating proteins in serum, the protein content after fractionation and concentration treatment is preferably less than 1% before treatment for proteins with high molecular weight and high content such as serum albumin and immunoglobulin. If high-molecular-weight and high-content proteins remain in the protein solution after fractionation at 1% or more before treatment, detection of specific proteins may be hindered when analyzing by mass spectrometry or two-dimensional electrophoresis. Therefore, it is not preferable. On the other hand, in a sample in which the high molecular weight and high content protein is less than 1% before processing, it becomes possible to detect many specific proteins by mass spectrometry and two-dimensional electrophoresis.

Furthermore, in the protein fractionation method of the present invention, the solution containing the protein is preferably a body fluid containing the protein. Thereby, in particular, proteins contained in body fluids such as blood and urine can be efficiently fractionated, and specific proteins contained in the fractionated solution can be removed or recovered.
The “solution containing protein” in the present invention refers to all liquids in which protein is dissolved, and the type, content and concentration of the protein contained therein are not particularly limited. Also, the type of solvent is not particularly limited as long as the protein can be dissolved. In addition, the “protein-containing body fluid” in the present invention refers to a fluid constituting a general living body, such as blood, plasma, serum, lymph fluid, urine, saliva, bone marrow fluid, ascites, pleural effusion, sweat, Examples include tears and breast milk.
The protein fractionation device of the present invention preferably recovers “disease marker protein”. The disease marker protein refers to a protein whose expression level or modification state varies greatly depending on the disease.

  The protein fractionation method of the present invention is a protein fractionation device comprising a purification module for purifying a protein in a solution treated by a fractionation module filled with a hollow fiber membrane, the purification module Using a protein fractionation device characterized by having a function of selectively adsorbing a specific protein in the solution, the specific protein is removed or recovered from the solution containing the protein. As a result, proteins contained in the solution can be efficiently fractionated, and specific proteins can be removed or recovered from the fractionated proteins with high efficiency.

  Furthermore, in the protein fractionation method of the present invention, preferably, the solution containing the protein is fractionated and collected with a difference in molecular weight. Thereby, the protein contained in the body fluid can be efficiently fractionated using the difference in molecular weight, and the specific protein contained in the fractionated solution can be removed or recovered.

  As described above, the protein fractionation device of the present invention can efficiently recover the target protein from the solution containing the protein. Therefore, by using the protein fractionation device of the present invention, for example, efficiently recovering target proteins from proteins contained in serum and plasma, and analyzing these proteins by mass spectrometry, two-dimensional electrophoresis, etc. It is now possible to quantitatively compare the expression level of disease marker protein contained in the serum and plasma of patients with that of healthy subjects, and to compare the degree to which the disease marker protein has undergone post-translational modifications. It is possible to know increase / decrease in protein expression level and change in post-translational modification state due to disease. Therefore, the protein fractionation device of the present invention is particularly useful for identification of disease-related proteins and creation of new drug discovery targets.

  Hereinafter, although an Example and a comparative example demonstrate in detail, this invention is not limited to these.

Example 1
An empty column with an inner diameter of 4 mm and a length of 10 mm was filled with 100 μL of phosphoprotein recovery beads (beads attached to PhosphoProtein Purification Kit manufactured by Qiagen) to prepare a purification module A. A schematic diagram of the purification module A is shown in FIG.

  The resin-bonded portions at both ends of the hemodialyzer (Toray TS1.6ML) were cut to obtain a polysulfone hollow fiber membrane. The dimensions of the obtained hollow fiber membrane were an inner diameter of 200 μm and a film thickness of 40 μm. When the cross section of the portion through which the liquid permeated was observed, it had an asymmetric structure. 100 hollow fiber membranes were bundled, and both ends were fixed to a plastic tube module case with an epoxy-based potting agent so as not to block the hollow portion of the hollow fiber membrane, thereby producing a mini module. The mini-module has a diameter of about 8 mm and a length of about 13 cm. Like a general hollow fiber membrane dialyzer, there are two ports (circulation ports) and two dialysate ports for circulating a liquid in the hollow fiber. I have one. A fraction module was obtained by thoroughly washing the hollow fiber membrane of the produced mini-module and the inside of the module with distilled water.

  A fraction circulation circuit was produced using the fraction module and a silicon tube (made by ASONE) having an outer diameter of 4 mm and an inner diameter of 2 mm. A fixed-quantity liquid pump (manufactured by Tokyo Rika Kikai Co., Ltd.) was installed in the middle of the fractional circulation circuit, allowing the liquid in the circuit to circulate. In addition, a port of a hemodialysis circuit (manufactured by Toray) was cut out and installed in a fractional circulation circuit, and a sample inlet was provided. The filtrate outlet of the fractionation module and the inlet of the purification module A were connected by a silicon tube. Between the fractionation module and the purification module A, a quantitative liquid feeding pump (hereinafter, liquid feeding pump B) was installed. In addition, a three-way valve was installed and connected to a gradient pump. A container capable of containing a washing solution and an elution buffer was connected to the tip of the gradient forming pump. The purification module A is connected, and the outlet of the purification module A is divided into two channels using a three-way valve and a silicon tube. One end is directed to the reservoir, and the other end is the fractor collector (GE Health). (Care, F-950 type). Further, the reservoir and the inlet of the purification module A were connected by a fixed amount feeding pump and a silicon tube provided with a three-way valve to form a concentration circulation circuit. The fraction circulation circuit was filled with 50 mM ammonium bicarbonate buffer (pH 8.0) (hereinafter referred to as buffer A) to produce a protein fractionation device (hereinafter referred to as fractionation device A). A schematic diagram of the fractionation device A is shown in FIG.

Example 2
An empty column having an inner diameter of 4 mm and a length of 10 mm was filled with 100 μL of Con A Sepharose 4B beads (manufactured by GE Healthcare) to produce a column similar to that of Example 1, and designated as purification module B. Then, a circuit was prepared in the same manner as in Example 1, and a buffer solution A was filled in the fractionation circulation circuit to produce a protein fractionation device (hereinafter referred to as fractionation device B).

Example 3
An empty column having an inner diameter of 4 mm and a length of 10 mm was filled with 100 μL of Q Sepharose 4B beads (manufactured by GE Healthcare) to produce a column similar to that of Example 1, and designated as purification module C.

  Purification module A and purification module C are connected in parallel using a silicon tube and a three-way valve, a circuit is created in the same manner as in Experimental Examples 1 and 2, buffer A is filled in the fractionation circulation circuit, and protein fractionation is performed. A device was produced (hereinafter referred to as fractionation device C). A schematic diagram of the fractionation device C is shown in FIG.

Example 4
A flat plate made of polymethyl methacrylate (PMMA) (load deflection temperature: 92 ° C.) was processed into a size of 79 × 23 × 2 mm to form a flow path having a width of 1 mm × height of 1 mm × length of 40 mm. 20 μl of POROS 20 HQ beads (manufactured by Applied Biosystems) were filled in the flow path, and the upper plate was further bonded to obtain a purification module D. A schematic diagram of the purification module D is shown in FIG.

  Purification module A and purification module D are connected in parallel using a silicon tube and a three-way valve, a circuit is created in the same manner as in Experimental Examples 1 and 2, buffer A is filled in the fractionation circulation circuit, and protein fractionation is performed. A device was produced (hereinafter referred to as a fractionation device D). A schematic diagram of the fractionation device D is shown in FIG.

Example 5
The purification module A and the purification module C were connected in parallel using a silicon tube and a three-way valve, and a circuit was prepared in the same manner as in Experimental Examples 1 and 2. However, the circuit was changed as follows. First, a silicon tube was connected to the filtrate outlet of the fractionation module so that the filtrate could be collected in the reservoir. In order to circulate the solution in the container to the purification module, the following purification circuit was set up. The end of the silicon tube connected to the inlet side of both purification modules was able to be immersed in the solution in the reservoir, and the liquid could be fed by installing a metered liquid feed pump. In addition, a silicon tube connected to the outlet side of both purification modules was also installed so that the solution that passed through it entered the reservoir. A three-way valve was installed in the middle of this silicon tube circuit, and the solution could be collected in the fraction collector by switching it. The fractionation circuit was filled with buffer A to produce a protein fractionation device (hereinafter referred to as fractionation device E). A schematic diagram of the fractionation device E is shown in FIG.

Example 6
An empty column having an inner diameter of 4 mm and a length of 10 mm was filled with 100 μL of OASIS (registered trademark) HLB (Waters), which is a reverse phase resin, to produce a column similar to that of Example 1, and designated as purification module E.

  The purification module C and the purification module E were connected in series using a silicon tube and a three-way valve, and a circuit was prepared in the same manner as in Experimental Examples 1 and 2. A three-way valve was installed between the fractionation module and the purification module A and connected to a gradient forming pump (hereinafter referred to as gradient forming pump A). At the end of the gradient forming pump, a container capable of containing an elution start buffer and an elution buffer (hereinafter, elution start buffer A and elution buffer A, respectively) was connected. Furthermore, a three-way valve was installed between the outlet of the purification module C and the inlet of the purification module E, and connected to a gradient forming pump (hereinafter referred to as gradient forming pump B).

 At the end of the gradient forming pump, a container capable of containing an elution start buffer and an elution buffer (hereinafter, elution start buffer B and elution buffer B, respectively) was connected. The fractionation circuit was filled with buffer A to produce a protein fractionation device (hereinafter referred to as fractionation device F). A schematic diagram of the fractionation device F is shown in FIG.

Example 7
Two fraction modules A filled with 100 hollow fiber membranes described in Example 1 (hereinafter referred to as fraction module A1 and fraction module A2) and 40 hollow fiber membranes were filled in the same manner. One fraction module B was produced. About each module, the circulation circuit which a liquid passes through the hollow fiber membrane inner side was each produced using the silicon tube of outer diameter 4mm and inner diameter 2mm. Furthermore, the outlet outside the hollow fiber membrane of the fractionation module A1 and the inlet inside the hollow fiber membrane of the fractionation module A2, and the outlet outside the hollow fiber membrane of the fractionation module A2 and the inlet inside the fractionation module B hollow fiber membrane. Each was connected with a silicon tube. Furthermore, a port for hemodialysis (manufactured by Toray) was cut out and installed at the inlet of the fractionation section, and an inlet for sample and buffer was provided. In this way, a fractionation part A was produced. The purification module A and the purification module C were connected in parallel with a silicon tube, and the filtrate outlet of the fractionation unit and the inlets of both purification modules were connected with a silicon tube. A quantitative liquid feeding pump was installed between the fractionation unit and both purification modules. In addition, a silicon tube was connected to the outlets of both modules, and the tip portion was directed to the reservoir A. In addition, a circuit for circulating the liquid in the reservoir A to the purification module with a silicon tube was provided to form a concentration circulation circuit. At this time, a metering pump was installed in the middle of the circuit, and one end was connected to the reservoir A and the other end was connected to a three-way valve installed in the circuit. The circuit of the fractionation part was filled with buffer A to produce a protein fractionation device (hereinafter referred to as fractionation device G). A schematic diagram of the fractionation device G is shown in FIG.

Example 8
1 mL of human commercially available serum (Sigma, Cat. H1388) is centrifuged to precipitate insoluble matter, and the supernatant is filtered using a MILLEX-GL filter (Millipore) having a pore size of 0.22 μm, and 4 times with buffer A. Diluted (hereinafter referred to as diluted serum A). 4.5 mL of diluted serum was taken into a 5 mL syringe (manufactured by Terumo), a silicon tube was attached to the tip thereof, and the microsyringe pump (manufactured by KD Scientific) was set. In addition, take 50 mM ammonium bicarbonate buffer (pH 8.0) in a 50 mL syringe (made by Nipro), fill the diluted serum up to the tip of the needle, confirm that no air bubbles are mixed in, and then remove the needle tip from the protein component. The injection port provided in the circuit of the image device A was stabbed and connected. The pump A is operated to circulate the buffer A filled in the circulation circuit at 5 mL / min, and then the syringe pump is operated to push out the diluted serum at 0.2 mL / min. When activated, the filtrate of the fractionation module was fed into the purification circuit at 0.2 mL / min and circulated for 1 hour. All the pumps were stopped, the three-way valve was switched, and the protein adsorbed on the purification module A was eluted by a linear gradient of the elution start buffer and elution buffer. 2 ml of buffer A was flowed as the elution start buffer, and 2 ml of 50 mM sodium phosphate buffer (pH 9.0) was flowed into the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. The amount of phosphorylated protein in each fraction was measured by enzyme immunoassay (ELISA), and the recovery rate was calculated by comparing with the content before treatment. As a result, the recovery rate of phosphorylated protein was 30%. In addition, the protein contained in the solution before the treatment and the collected solution is enzymatically digested with Sequencing Grade Modified Trypsin (Promega), and the eluted protein solution using an ESI-Q-TOF type mass spectrometer (manufactured by Micromass) An attempt was made to detect the phosphorylated protein in the inner surface. As a result, phosphorylated proteins could not be detected from the solution before treatment, whereas four types of phosphorylated proteins could be detected. Therefore, the protein to be recovered could be obtained selectively and with a high recovery rate.

Example 9
In the same manner as in Example 8, a syringe containing diluted serum A was set in a syringe pump and connected to the circuit of the protein fractionation device B. As in Example 6, serum A was introduced into the circuit and circulated for 1 hour. Thereafter, all the pumps were stopped, the three-way valve was switched, and the protein adsorbed on the purification module B was eluted by a linear gradient gradient between the elution start buffer and the elution buffer. 2 ml of buffer A was flowed as the elution start buffer, and 2 ml of 50 mM Tris-buffer (pH 3.5) containing 200 mM alpha-mannose was flowed into the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. The mass of glycoprotein in each fraction was measured by enzyme immunoassay (ELISA), and the recovery rate was calculated by comparing with the content before treatment. As a result, the recovery rate of glycoprotein was 30%. In addition, the protein contained in the solution before the treatment and the collected solution is enzymatically digested with Sequencing Grade Modified Trypsin (Promega), and the eluted protein solution using an ESI-Q-TOF type mass spectrometer (manufactured by Micromass) An attempt was made to detect the glycoproteins in the membrane. As a result, glycoproteins could not be detected from the solution before the treatment, but 6 types of glycoproteins could be detected. Therefore, the protein to be recovered could be obtained selectively and with a high recovery rate.

Example 10
In the same manner as in Example 8, a syringe containing diluted serum A was set in a syringe pump and connected to the circuit of the protein fractionation device C. In the same manner as in Example 5, serum A was introduced into the circuit, and the flow path was connected to the purification module A and circulated for the first hour, and the flow path was connected to the purification module C and circulated for the next hour. . Thereafter, all the pumps were stopped, the three-way valve was switched, and the protein adsorbed on the purification module A was first eluted by a linear gradient gradient between the elution start buffer and the elution buffer. 2 ml of buffer A was flowed as the elution start buffer, and 2 ml of 50 mM sodium phosphate buffer (pH 9.0) was flowed into the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. Next, the three-way valve was further switched, and the protein adsorbed on the purification module B was eluted with a linear gradient gradient between the elution start buffer and the elution buffer. At this time, 2 ml of buffer A was used as the elution start buffer, and 2 mL of 50 mM ammonium formate buffer (pH 3.5) containing 100 mM sodium chloride was passed through the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. The amount of phosphorylated protein in each fraction collected from the purification module A was measured by enzyme immunoassay (ELISA), and the recovery rate was calculated by comparing with the content before treatment. As a result, the recovery rate of phosphorylated protein was 30%. Similarly, the concentration of β2MG, IL-6 and IL-8 contained in the HSA and the protein to be collected in the fraction eluted from the purification module C is measured to determine the content and compared with the content before treatment. The recovery rate was calculated. As a result, the recovery rates of HSA were 0.04%, while the recovery rates of β2MG, IL-6, and IL-8 were 30%, 40%, and 43%. Therefore, the protein to be recovered could be obtained selectively and with a high recovery rate.

Example 11
In the same manner as in Example 8, a syringe containing diluted serum A was set in a syringe pump and connected to the circuit of the protein fractionation device D. Serum A is introduced into the circuit in the same manner as in Example 5, and the flow path is connected to the purification module A for the first hour and circulated. Circulation was performed at 0.01 mL / min. Thereafter, all the pumps were stopped, the three-way valve was switched, and the protein adsorbed on the purification module A was first eluted by a linear gradient gradient between the elution start buffer and the elution buffer. 2 ml of buffer A was flowed as the elution start buffer, and 2 ml of 50 mM sodium phosphate buffer (pH 9.0) was flowed into the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. Next, the three-way valve was further switched, and the protein adsorbed on the purification module D was eluted with a linear gradient gradient between the elution start buffer and the elution buffer. At this time, 2 ml of buffer A was used as the elution start buffer, and 2 mL of 50 mM ammonium formate buffer (pH 3.5) containing 100 mM sodium chloride was passed through the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. The amount of phosphorylated protein in each fraction collected from the purification module A was measured by enzyme immunoassay (ELISA), and the recovery rate was calculated by comparing with the content before treatment. As a result, the recovery rate of phosphorylated protein was 30%. Similarly, the concentration of β2MG, IL-6, and IL-8 contained in the HSA and the protein to be recovered in the fraction eluted from the purification module D is measured to determine the content, and compared with the content before treatment. The recovery rate was calculated. As a result, the recovery rates of HSA were 0.02%, while the recovery rates of β2MG, IL-6, and IL-8 were 20%, 23%, and 20%. Therefore, the protein to be recovered could be obtained selectively and with a high recovery rate.

Example 12
In the same manner as in Example 8, a syringe containing diluted serum A was set in a syringe pump and connected to the circuit of the protein fractionation device E. In the same manner as in Example 5, serum A was introduced into the circuit, and the flow path was connected to the purification module A and circulated for the first hour, and the flow path was connected to the purification module C and circulated for the next hour. . Thereafter, all the pumps were stopped, the three-way valve was switched, and the protein adsorbed on the purification module A was first eluted by a linear gradient gradient between the elution start buffer and the elution buffer. 2 ml of buffer A was flowed as the elution start buffer, and 2 ml of 50 mM sodium phosphate buffer (pH 9.0) was flowed into the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. Next, the three-way valve was further switched, and the protein adsorbed on the purification module C was eluted with a linear gradient gradient between the elution start buffer and the elution buffer. At this time, 2 ml of buffer A was used as the elution start buffer, and 2 mL of 50 mM ammonium formate buffer (pH 3.5) containing 100 mM sodium chloride was passed through the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. The amount of phosphorylated protein in each fraction collected from the purification module A was measured by enzyme immunoassay (ELISA), and the recovery rate was calculated by comparing with the content before treatment. As a result, the recovery rate of phosphorylated protein was 30%. Similarly, the concentration of β2MG, IL-6 and IL-8 contained in the HSA and the protein to be collected in the fraction eluted from the purification module C is measured to determine the content and compared with the content before treatment. The recovery rate was calculated. As a result, the recovery rates of HSA were 0.04%, while the recovery rates of β2MG, IL-6, and IL-8 were 30%, 40%, and 43%. Therefore, the protein to be recovered could be obtained selectively and with a high recovery rate.

Example 13
In the same manner as in Example 8, the syringe containing the diluted serum A was set in the syringe pump and connected to the circuit of the protein fractionation device F. As in Example 5, serum A was introduced into the circuit and circulated for 1 hour. After that, all pumps are stopped, the three-way valve is switched, the purification module E is washed with the buffer B for elution start, and then the purification module E is first subjected to a linear gradient gradient between the buffer B for elution start and the buffer B for elution. The protein adsorbed on E was eluted. Elution buffer B is 2 ml of 10 mM ammonium acetate buffer (pH 4.5), and elution buffer B is 2 mL of 10 mM ammonium acetate buffer (pH 4.5) containing 70% acetonitrile (v / v). It was. The eluted protein solution was collected every 100 μl using a fraction collector. Next, the three-way valve is further switched, and the protein adsorbed on the purification module C is partially eluted by feeding the elution start buffer A and the elution buffer A at a mixing ratio of 1: 9, Adsorbed to purification module E. At this time, 10 mM ammonium acetate buffer (pH 4.5) was used as buffer A for elution start, and 500 mM ammonium acetate buffer (pH 4.5) was used as buffer A for elution. After switching the three-way valve and washing the purification module E with the elution start buffer B, the protein adsorbed on the purification module E is eluted with a linear gradient gradient between the elution start buffer B and the elution buffer B, Recovered using a fraction collector. Next, the three-way valve is further switched, and the protein adsorbed on the purification module C is partially eluted by feeding the elution start buffer A and the elution buffer A at a mixing ratio of 1: 4, Adsorbed to purification module E. In the same manner as described above, after switching the three-way valve and washing the purification module E with the elution start buffer B, the protein adsorbed on the purification module E by the linear gradient gradient of the elution start buffer B and the elution buffer B. Was eluted and collected using a fraction collector. Further, the three-way valve was switched and only the elution buffer A was fed, so that all the proteins adsorbed on the purification module C were completely eluted and adsorbed on the purification module E. Further, after switching the valve and washing the purification module E with the elution start buffer B, the protein adsorbed on the purification module E is eluted with a linear gradient gradient between the elution start buffer B and the elution buffer B, Recovered using a fraction collector.

  The concentration of β2MG, IL-6, and IL-8 contained in each fraction collected by enzyme immunoassay (ELISA) was determined by measuring the concentrations of HSA and β2MG, IL-6, and IL-8. The recovery rate was calculated by comparison. As a result, the recovery rates of HSA were below the detection limit, whereas the recovery rates of β2MG, IL-6, and IL-8 were 18%, 20%, and 17%.

Example 14
In the same manner as in Example 8, a syringe containing diluted serum A was set in a syringe pump and connected to the circuit of the protein fractionation device G. In the same manner as in Example 5, serum A was introduced into the circuit, and the flow path was connected to the purification module A and circulated for the first hour, and the flow path was connected to the purification module C and circulated for the next hour. . Thereafter, all the pumps were stopped, the three-way valve was switched, and the protein adsorbed on the purification module A was first eluted by a linear gradient gradient between the elution start buffer and the elution buffer. 2 ml of buffer A was flowed as the elution start buffer, and 2 ml of 50 mM sodium phosphate buffer (pH 9.0) was flowed into the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. Next, the three-way valve was further switched, and the protein adsorbed on the purification module C was eluted with a linear gradient gradient between the elution start buffer and the elution buffer. At this time, 2 ml of buffer A was used as the elution start buffer, and 2 mL of 50 mM ammonium formate buffer (pH 3.5) containing 100 mM sodium chloride was passed through the elution buffer. The eluted protein solution was collected every 100 μl using a fraction collector. The amount of phosphorylated protein in each fraction collected from the purification module A was measured by enzyme immunoassay (ELISA), and the recovery rate was calculated by comparing with the content before treatment. As a result, the recovery rate of phosphorylated protein was 30%. Similarly, the concentration of β2MG, IL-6 and IL-8 contained in the HSA and the protein to be collected in the fraction eluted from the purification module C is measured to determine the content and compared with the content before treatment. The recovery rate was calculated. As a result, the recovery rates of β2MG, IL-6, and IL-8 were 24%, 32%, and 31% while being below the HSA detection limit. Therefore, the protein to be recovered could be obtained selectively and with a high recovery rate.

Comparative Example 1
A purification circuit B was created in which the inlet and outlet of the purification module A were connected by a silicon tube. The ends of the silicon tubes on both the inlet side and the outlet side can be immersed in the solution in the reservoir, and the liquid can be fed by installing a metered liquid feeding pump. Furthermore, a three-way valve was installed so that a syringe for feeding the elution start buffer and elution buffer could be attached. Further, a silicon tube was connected to the outlet side of the purification module A using a three-way valve, and the end portion was directed to the recovery unit. Diluted serum A was placed in the reservoir, and the pump was turned on to circulate in the purification circuit B for 2 hours. Thereafter, the pump was stopped and 0.5 mL of 50 mM sodium phosphate buffer (pH 9.0) was passed through the purification module A to elute the phosphorylated protein adsorbed on the purification module A. In the same manner as in Example 7, the concentration of HSA contained in the protein to be removed in this solution was measured by enzyme immunoassay (ELISA) to determine the content, and the recovery rate was compared with the content before treatment. Calculated. As a result, the recovery rate of HSA was 0.05%. In addition, the recovered protein solution is enzymatically digested with Sequencing Grade Modified Trypsin (Promega), and phosphorous in the eluted protein solution is obtained by a standard method using an ESI-Q-TOF type mass spectrometer (manufactured by Micromass). Attempts were made to detect oxidized proteins. However, phosphorylated proteins were not detected, and most were albumin signals.

Sectional drawing which showed the one aspect | mode of the refinement | purification module in this invention is represented. The conceptual diagram which showed the one aspect | mode of the protein fractionation device in this invention is represented. The conceptual diagram which showed the one aspect | mode of the protein fractionation device in this invention is represented. Sectional drawing which showed the one aspect | mode of the refinement | purification module in this invention is represented. The conceptual diagram which showed the one aspect | mode of the protein fractionation device in this invention is represented. The conceptual diagram which showed the one aspect | mode of the protein fractionation device in this invention is represented. The conceptual diagram which showed the one aspect | mode of the protein fractionation device in this invention is represented. The conceptual diagram which showed the one aspect | mode of the protein fractionation device in this invention is represented.

Explanation of symbols

101 Filled beads 102 Filter 201a Syringe 201b Sampled syringe 201b Syringe 202a, 202b, 202c, 202d containing 50 mM ammonium hydrogen carbonate buffer (pH 8.0) Three-way valve 203 Sample inlet 204 Fraction circulation channel 205a, 205b, 205c Liquid feeding pump 206 Fractionation module 207 Purification module 208 Reservoir 209 Fraction collector 210 Gradient solution pump 211a, 211b Elution buffer, Elution start buffer 301a Syringe containing sample 301b Syringe 302a containing 50 mM ammonium bicarbonate buffer (pH 8.0) 302b, 302c, 302d, 302e, 302f Three-way valve 303 Sample inlet 304 Fraction circulation channel 305a, 305b, 305c Liquid feed pump 306 min Module 307 Purification module 308 Purification module 309 Reservoir 310 Fraction collector 311 Gradient solution pump 312a, 312b Elution buffer, Elution start buffer 401 PEEK tube 402 Screw connector 403 Upper plate 404 Lower plate 405 Filled beads 406 Filter 501a Sample Syringe 501b Syringe 502a, 502b, 502c, 502d, 502f Three-way valve 503 Sample inlet 504 Fraction circulation flow path 505a, 505b, 505c Feed pump 506 Fractionation module 507 Purification module 508 Purification module 509 Reservoir 510 Fraction collector 511 Gradient solution pump 512a, 512b Loose for elution Solution, elution start buffer 401a syringe with sample 601a syringe with sample 601b syringe with 50mM ammonium bicarbonate buffer (pH 8.0) 602a, 602b, 602c, 602d, 602e three-way valve 603 sample inlet 604 fraction circulation channel 605a, 605b Feed pump 606 Fractionation module 607 Purification module 608 Purification module 609 Reservoir 610 Fraction collector 611 Gradient feed pump 612a, 612b Elution buffer, Elution start buffer 701a Sampled syringe 701b 50 mM ammonium hydrogen carbonate buffer (PH 8.0) Syringe 702a, 702b, 702c, 702d, 702e Three-way valve 703 Sample inlet 704 Fraction circulation channel 705a, 705b, 70 c Liquid feed pump 706 Fractionation module 707 Purification module 708 Purification module 709 Reservoir 710 Fraction collector 711a, 711b Gradient liquid feed pump 712a, 712b, 712c, 712d Elution buffer, elution start buffer 801a Sampled syringe 801b 50 mM carbonic acid Syringes 802a, 802b, 802c, 802d, 802e, 802f Three-way valves 803a, 803b, 803c Sample inlets 804a, 804b, 804c, 804d, 804e Feed pumps 805a, 805b, 805c Drawing module 806 Purification module 807 Purification module 808 Reservoir 809 Fraction collector 810 Gradient liquid feed pump 811a, 81 b elution buffer, the elution starting buffer

Claims (11)

A protein fractionation device comprising a purification module for recovering a protein in a solution processed by a fractionation module filled with a hollow fiber membrane, wherein the purification module selectively selects a specific protein in the solution. A protein fractionation device characterized by having a function of adsorbing and recovering. The protein fractionation device according to claim 1, wherein a solution containing the protein treated by the fractionation module is circulated to the purification module for treatment. The protein fractionation device according to claim 1 or 2, wherein the fractionation module and the purification module are connected by a solution channel and can be operated continuously. 1 from the group consisting of metal chelate resin, protein immobilization resin, antibody immobilization resin, lectin immobilization resin, group specific affinity resin, and hydrophobic compound immobilization resin, ion exchange resin, reverse phase resin The protein fractionation device according to any one of claims 1 to 3, comprising a resin selected from at least one type. The protein fractionation device according to any one of claims 1 to 4, comprising a plurality of purification modules containing different resins. All the purification modules are connected with respect to the solution flow path, The protein fractionation device in any one of Claims 1-5 characterized by the above-mentioned. The protein fractionation device according to any one of claims 1 to 6, wherein a specific protein is collected as a fraction. The protein fractionation device according to any one of claims 1 to 7, wherein 20% or more of the specific protein is recovered. The protein fractionation device according to any one of claims 1 to 8, wherein the solution containing the protein is a body fluid containing the protein. The protein fractionation device according to any one of claims 1 to 9, wherein the specific protein is a disease marker protein. A method for fractionating a protein-containing solution using the protein fractionation device according to claim 1 based on a difference in molecular weight.

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