JP5780519B2 - Method for producing porous membrane having affinity function and method for separating and purifying protein - Google Patents

Description

  The present invention relates to a method for producing a porous membrane and a method for separating and purifying proteins.

  In recent years, in various research and development and analysis sites, high throughput has been promoted to enable simultaneous processing of a wide variety of samples and information for the purpose of labor saving, sample weight reduction, and data refinement. It has been. For example, in the field of antibody drugs based on in vivo antigen-antibody reactions, screening for selecting a large number of proteins is performed, and at that time, it is necessary to purify various proteins at once. Antibody drugs have attracted particular attention in recent years because they selectively act on affected areas and have few side effects. Usually, recombinant proteins are produced using E. coli or animal cells as hosts, and at this time, a large number of impurity proteins are produced simultaneously. Therefore, it is necessary to crush the host cell and elute the target protein, and then to separate the cell crushed material from the target protein by centrifugation or a filter. Thereafter, the liquid containing the target protein and the impurity protein is treated by various types of chromatography to separate and purify the target protein. The types of chromatography are gel filtration chromatography using separation by sieving effect, adsorption chromatography using the difference in protein adsorption capacity (ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography). Chromatography, reverse phase chromatography).

  In recent years, affinity chromatography that separates and purifies proteins using a carrier having a ligand that specifically and reversibly adsorbs to a specific protein has attracted attention. This is because affinity chromatography has higher purification efficiency and recovery rate than other chromatography, and has the advantage that a large amount of sample can be processed at one time.

  For example, in Non-Patent Document 1, an affinity chromatography bead in which glutathione is introduced as an affinity ligand to agarose beads as a carrier and a glutathione-S-transferase (GST) fusion protein are separated and purified using the affinity beads. A method is disclosed.

  Affinity chromatography using a porous membrane as a carrier is also known. For example, Patent Document 1 discloses a method in which a porous crosslinked cellulose membrane is prepared in advance as a carrier, and an affinity ligand composed of a quaternary ammonium group and a diethylaminoatyl group is introduced into this by a chemical reaction. Non-Patent Document 2 discloses membrane-type affinity chromatography obtained by preparing a regenerated cellulose porous membrane in advance and immobilizing glutathione on this by a chemical reaction.

Special table 2009-503160

GE Healthcare Co., Ltd. Product Catalog, 28-9622-84AA, December 2009, [online], [Search August 15, 2011], Internet <URL: http://www.gelifesciences.co.jp/ catalog / pdf / 28962284.pdf> Biochimica et Biophysica Acta, 1994, 1205, p. 29-38

  In the case of affinity chromatography using beads as a carrier, as disclosed in Non-Patent Document 1, before treatment of the treatment liquid containing the target protein by chromatography, the cells are separated from the treatment liquid by centrifugation or filtration. It is necessary to remove suspended substances such as crushed material. Therefore, there are many steps, and a large amount of liquid to be processed containing the target protein is required. In addition, when high-speed processing is performed, channeling occurs in which the liquid to be processed passes between the beads, and the target protein permeates the filtrate without being adsorbed by the ligand.

  On the other hand, in the case of affinity chromatography using a porous membrane as a carrier, when the treatment liquid containing the target protein, impurity protein and cell lysate is treated, the crushed cell and the target The target protein and the impurity protein can be separated by the affinity ligand while separating the protein and the impurity protein. That is, according to affinity chromatography using a porous membrane as a carrier, separation and purification can be performed in a one-step process. Furthermore, since the liquid containing the target protein is sent by forced flow to the ligand supported in the pores, the contact efficiency between the ligand and the target protein is high, and high-speed processing is possible. There is also an advantage that scale-up is easy.

  However, since affinity chromatography using a porous membrane as a carrier has been conventionally produced by a method in which an affinity ligand is introduced into a porous membrane by a chemical reaction, the steps required for the production are complicated. In order to reduce the cost, development of a method for producing affinity chromatography using a porous membrane as a carrier by a simpler process is required.

  An object of the present invention is to provide a method that makes it possible to produce a porous membrane having an affinity function suitable for protein separation and purification by a simple process. Another object of the present invention is to provide a protein separation and purification method using a porous membrane obtainable by such a method.

  As a result of intensive studies, the present inventors have obtained a porous membrane from a polymer solution (membrane forming stock solution) containing an organic compound having a hydrophobic group bound to glutathione, which is an affinity ligand, using a phase separation method. By forming it, it was found that a porous membrane having an affinity function with high separation accuracy can be produced by a simple process, and the present invention was completed.

That is, the present invention relates to the following method.
(1) obtaining a porous membrane precursor formed by phase-separating a polymer solution containing a polymer, an organic liquid dissolving the polymer, and an organic compound having a hydrophobic group bonded to glutathione and glutathione; And removing the organic liquid from the porous membrane precursor to form a porous membrane containing the polymer and the organic compound.
(2) The hydrophobic group is selected from the group consisting of an alkyl group, an alkylene group, an aryl group, and a group represented by R (C═O) —, wherein R is a saturated aliphatic group or an unsaturated aliphatic group. The method of (1), wherein
(3) The method of (1) or (2), wherein the hydrophobic group is a hexadecyl group.
(4) The method according to any one of (1) to (3), wherein the polymer is a cellulose derivative.
(5) A stock solution containing glutathione-S-transferase fusion protein is filtered through a porous membrane obtainable by the method according to any one of (1) to (4), and glutathione-S-transferase fusion protein is applied to the porous membrane. A method for separating and purifying a glutathione-S-transferase fusion protein, which comprises the step of adsorbing.

  According to the present invention, a porous membrane having an affinity function suitable for protein separation and purification can be produced by a simple process. Since the process is simple, the porous membrane can be provided at low cost. The present invention also provides a protein separation and purification method using the porous membrane produced by the above method. According to the separation and purification method of the present invention, separation and purification in a single stage process is possible. In addition, since the liquid containing the target protein is sent by forced flow to the ligand supported in the pores, the contact efficiency between the ligand and the target protein is high, high-speed processing is possible, and scale-up is easy. .

  According to the conventional method of immobilizing affinity ligands on the surface of the porous membrane by a chemical reaction, it is difficult to introduce the ligand into the pores inside the porous membrane. There is a problem that the separation performance of the protein is lowered. On the other hand, according to the method of the present invention, the density of the ligand density on the surface of the porous membrane hardly occurs. In addition, when an affinity ligand is introduced by a chemical reaction as in the prior art, there is a problem that a porous membrane itself formed from an organic material is deteriorated by a chemical such as an alkali, an acid, or an oxidizing agent to be used. Thus, the method of the present invention can avoid such a problem. The production cost of a porous film using an organic material is generally lower than that of a porous film using an inorganic material.

It is a schematic diagram showing one embodiment of a porous membrane. It is a graph which shows the result of having isolate | separated and refine | purified the target protein from the to-be-processed liquid containing a target protein using a porous membrane. It is a graph which shows the result of having separated and refine | purified impurity protein from the to-be-processed liquid containing impurity protein using a porous membrane. It is a graph which shows the result of having separated and refine | purified the target protein from the to-be-processed liquid containing a target protein and impurity protein using a porous membrane.

  Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be variously modified within the scope of the gist thereof.

[Method for producing porous film having affinity function]
The method according to the present embodiment includes a polymer, an organic liquid that dissolves the polymer, and an organic compound having an affinity ligand glutathione and a hydrophobic group bonded to glutathione (hereinafter sometimes referred to as “ligand compound”). A step of obtaining a porous membrane precursor formed by phase-separating a polymer solution containing the solution, and a step of forming a porous membrane containing a polymer and a ligand compound by removing an organic liquid from the porous membrane precursor obtained by phase separation With.

  The polymer functions as a base material that forms pores in the porous membrane. The polymer is not particularly limited, and examples thereof include polysulfone, polyethersulfone, polyacrylonitrile, cellulose acetate, cellulose propionate, cellulose butyrate, polyvinylidene fluoride, polyethylene, polypropylene, polytetrafluoroethylene, polycarbonate, polyester, polystyrene and poly It is at least one selected from the group consisting of methyl methacrylate. Among these, cellulose derivatives such as cellulose acetate, cellulose propionate, and cellulose butyrate are preferable. These cellulose derivatives have high hydrophilicity and can suppress nonspecific adsorption of impurity proteins to the porous membrane. The molecular weight of the polymer can be appropriately selected in consideration of the film forming property and strength. Different molecular weight polymers may be blended.

  The organic liquid that dissolves the polymer dissolves the polymer at normal temperature (for example, 20 ° C.) or high temperature (for example, 50 to 300 ° C.). A homogeneous polymer solution is formed by dissolving the polymer in this organic liquid.

  When the polymer is polysulfone or polyethersulfone, the organic liquid is selected from N-methyl-2-pyrrolidone, N, N-dimethylformamide, N, N-dimethylacetamide and the like. When the polymer is polyacrylonitrile, the organic liquid is selected from N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone, and the like. When the polymer is a cellulose derivative such as cellulose acetate, cellulose propionate and cellulose butyrate, the organic liquid when non-solvent induced phase separation is utilized is acetone, dioxane, dimethyl sulfoxide, N, N-dimethylacetamide, N The organic liquid (latent solvent) selected from methyl-2-pyrrolidone and N, N-dimethylformamide and the like when the thermally induced phase separation method is used is triethylene glycol, 2-methyl-2,4-pentane It is selected from diol and 2-ethyl-1,3-hexanediol. When the polymer is polyvinylidene fluoride, N-methyl-2-pyrrolidone, N, N-dimethylformamide, N, N-dimethylacetamide, dimethylsulfoxide, etc. when non-solvent induced phase separation is used The organic liquid (latent solvent) when the thermally induced phase separation method is used is dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dicyclohexyl phthalate, diheptyl phthalate, dioctyl phthalate and diphthalate phthalate Phthalates such as (2-ethylhexyl); ketones such as γ-butyrolactone, ethylene carbonate, propylene carbonate, cyclohexanone, acetophenone and isophorone; benzoates; and phosphates. When the polymer is polyethylene, the organic liquid is dibutyl phthalate, diheptyl phthalate, dioctyl phthalate, di (2-ethylhexyl) phthalate, diisodecyl phthalate and ditridecyl phthalate; liquid paraffin Paraffins such as; glycerin esters such as propylene glycol dicaprate and propylene glycol dioleate; adipic acid esters; and phosphates. These organic liquids may be used alone or in combination of two or more.

  The ligand compound has an affinity ligand that specifically and reversibly adsorbs to the target protein, and a hydrophobic group for immobilizing the affinity ligand on a porous membrane as a carrier. In the step of obtaining a porous membrane precursor, which will be described later, by separating the polymer solution into phases, the porous membrane is phase-separated into a concentrated phase having a high polymer concentration and a dilute phase having a lower polymer concentration than the concentrated phase. A precursor is formed. In the porous membrane precursor, the ligand compound is in a state in which the hydrophobic group having affinity with the polymer is directed to the rich phase side and the ligand is directed to the dilute phase side. As a result, the ligand is arranged on the pore surface in the porous membrane, and protein adsorption becomes possible.

  Hydrophobic groups have an affinity for the polymer. The hydrophobic group is represented by a linear or branched alkyl group, an alkylene group, an aryl group, and R (C═O) —, since the ligand can be easily introduced and the affinity with the polymer can be easily adjusted. R is preferably a group selected from the group consisting of a saturated aliphatic group or an unsaturated aliphatic group. The group represented by R (C═O) —, wherein R is a saturated aliphatic group or an unsaturated aliphatic group, may be derived from a saturated fatty acid or an unsaturated fatty acid. If these hydrophobic groups are too short, it is difficult to immobilize them in the polymer porous body. Therefore, the hydrophobic groups preferably have 10 to 30 carbon atoms.

  The affinity ligand is capable of specifically and reversibly adsorbing the target protein, and is a glutathione capable of specifically capturing a fusion protein (GST fusion protein) of the target protein and glutathione-S-transferase (GST).

  The porous membrane precursor can be obtained, for example, by phase-separating a polymer solution by a non-solvent induced phase separation method or a thermally induced phase separation method. In the non-solvent induced phase separation method, a liquid that is a non-solvent of the polymer is added to a solution in which the polymer is dissolved in an organic liquid to induce phase separation. In the thermally induced phase separation method, the polymer is dissolved in an organic liquid (latent solvent) that does not dissolve at room temperature but dissolves at a high temperature to form a uniform polymer solution, and then the temperature of the polymer solution is lowered to achieve phase separation. Induces. Crystalline polymers with excellent chemical stability and mechanical strength can be used, and the porosity of the obtained pores is high and the pore size distribution is sharp. It is preferable to form a porous membrane precursor by using it. When the porosity of the porous membrane is high, the substantial membrane area can be increased, so that the amount of protein that can be captured increases. Further, in a porous membrane having a sharp pore size distribution, the liquid to be treated flows uniformly into each pore, so that the amount of ligand that can be used before breakthrough increases. When the pore size distribution is large, the ligands on the large pores that are easy to flow are first adsorbed with proteins, and the lifetime of the membrane is determined by the time when the ligand breaks through.

  A porous membrane precursor is formed from a membrane-forming stock solution containing a polymer, an organic liquid, and a ligand compound. The film-forming stock solution may contain a high molecular weight organic substance such as polyethylene glycol and polyvinyl pyrrolidone, a low molecular weight organic substance such as tetraethylene glycol, or an inorganic substance such as lithium chloride, silica, calcium carbonate, and alumina as necessary. Good.

  When preparing the film-forming stock solution, all the components may be mixed simultaneously, or each component may be mixed sequentially. Alternatively, after some components are premixed, the remaining components may be mixed there. The mixing conditions (mixing temperature and mixing energy) and the atmosphere during mixing (in a vacuum or an inert gas atmosphere) can be appropriately selected. As an apparatus for mixing, a dissolution tank equipped with a facility capable of stirring and temperature adjustment may be used, and a single-screw or twin-screw extruder may be used. A defoaming operation may be performed in order to remove bubbles in the film-forming stock solution.

  The content of the polymer in the film-forming stock solution is preferably 10 to 60% by mass based on the total amount of the film-forming stock solution. When the content is 10% by mass or more, a porous film more excellent in terms of film forming property and mechanical strength can be obtained, and when the content is 60% by mass or less, a porous film having a high porosity is obtained. Therefore, a high protein adsorption amount can be achieved. The mass ratio of the ligand compound to the polymer is preferably 0.01 to 0.1. When the mass ratio is 0.01 or more, the porous membrane exhibits a particularly high protein capture performance. When the mass ratio is 0.1 or less, the porous membrane can be used even when a drug is used for protein separation and purification. Mechanical and chemical resistance is kept higher.

  When producing a flat membrane-like porous membrane, the membrane-forming stock solution can be formed into a flat membrane by extruding it from a T-die or applying it to a substrate such as a glass plate. At this time, the membrane-forming stock solution can be applied to the nonwoven fabric to obtain a composite membrane composed of the nonwoven fabric and the porous membrane. When producing a hollow fiber-like porous membrane, it can be extruded from a bicyclic spinneret and formed into a hollow shape. At this time, a non-solvent, a mixed solution of a solvent and a non-solvent, or a fluid such as air can be passed through the hollow portion. In addition, a hollow fiber membrane may be a composite membrane in which a film-forming stock solution is applied to a braid or the like.

  The gel-form film-forming stock solution formed into a flat film shape or a hollow fiber shape by the above-described method passes through an empty running part as necessary, and a non-solvent, a mixed solution of a non-solvent and a solvent, or a film-formation It is introduced into a tank filled with a liquid (for example, water) for cooling the stock solution, and solidified in a flat membrane state or hollow fiber state. A phase separation occurs with solidification to form a porous membrane precursor.

  From the porous membrane precursor, an organic liquid mainly contained in a dilute phase and optionally other additives are extracted and removed to obtain a porous membrane having a polymer phase that is a porous body forming pores. be able to. The solvent used for the extraction is not particularly limited as long as it does not substantially dissolve the polymer and dissolves the organic liquid. For example, water, alcohol, halogenated solvent, and the like may be appropriately selected depending on the polymer to be used. good. Here, the solvent that does not substantially dissolve the polymer is a solvent that dissolves 1% by mass or less of the polymer at the extraction temperature. The extraction method is not particularly limited as long as the organic liquid can be extracted from the porous membrane precursor. For example, the extraction may be immersed in an extraction solvent or the extraction solvent may be filtered. Further, heating may be performed during extraction, or extraction may be promoted by irradiation with ultrasonic waves or the like. Before and after the extraction, stretching and / or heat treatment may be performed to improve water permeability and strength properties.

  FIG. 1 is a schematic diagram showing an embodiment of a porous membrane. 1 includes a polymer (polymer phase) 1 forming pores 2 and a ligand compound (glutathione and an organic compound having a hydrophobic group bonded to glutathione) arranged on the surface of the polymer phase 1. 3). The ligand compound 3 has an affinity ligand (glutathione) 5 and a hydrophobic group 4 bonded thereto. Hydrophobic group 4 is immobilized on polymer phase 1. The affinity ligand 5 can specifically capture the GST fusion protein 6.

  The pore diameter of the porous membrane according to the present embodiment is preferably 10 nm to 10 μm, more preferably 50 nm to 5 μm. When the pore diameter is 10 nm or more, the filtration resistance of the membrane is low, higher filtration performance is obtained, and the permeability of impurity proteins is also high. In addition, when the pore diameter is 10 μm or less, the separation performance between the micron-order cell disrupted material and the protein is particularly excellent.

  The pore diameter can be measured by a method in which the size of the indicator substance having a blocking rate of 90% or more when the indicator substance having a known particle diameter is filtered through a porous membrane is used as the pore diameter. Specifically, by using monodisperse polystyrene particles as an indicator substance, a porous film having a pore diameter of 20 to 30 nm or more can be measured. Moreover, the measurement of the porous film which has a pore diameter of 20-30 nm or less can be performed by using protein as a parameter | index substance.

  The form of the porous membrane according to this embodiment may be a flat membrane shape or a hollow fiber shape. In the case of a flat membrane, the polymer may be a single polymer, or a composite membrane may be formed by supporting a porous membrane on a support such as a nonwoven fabric. The film thickness is preferably 5 μm to 1000 μm. When the film thickness is 5 μm or more, the contact between the protein-containing liquid and the affinity ligand on the membrane surface becomes large, and higher separation accuracy can be achieved. Moreover, if a film thickness is 1000 micrometers or less, the resistance at the time of a to-be-separated liquid permeate | transmits a film | membrane will be small, and higher water-permeable performance will be obtained.

  In the case of a hollow fiber-like porous membrane, the inner diameter (corresponding to the hollow portion) is preferably 100 μm to 5 mm. If the inner diameter is 100 μm or more, the pressure loss generated when the liquid flows through the hollow portion can be kept low, and if it is 5 mm or less, the film packing density per unit volume can be increased. It can be made compact.

  The film thickness of the hollow fiber-like porous membrane is preferably 5 μm to 1000 μm. If the film thickness is 5 μm or more, the contact between the protein-containing liquid and the affinity ligand on the surface of the membrane is increased, higher separation accuracy can be achieved, and sufficient internal pressure required for the internal pressure filtration hollow fiber porous membrane The burst strength can be obtained. In addition, when the film thickness is 1000 μm or less, the film packing density per unit volume can be increased, and downsizing is possible.

  The porosity of the porous film according to this embodiment is preferably 20% to 90%. If the porosity is 20% or more, particularly excellent water permeability and high adsorption capacity can be obtained, and if it is 90% or less, practical strength characteristics can be obtained.

  The porosity can be measured by a method in which the difference between the wet mass and the absolutely dry mass of a porous membrane impregnated with water in pores is divided by the membrane volume. In the case of a hollow fiber-like porous membrane, the porosity of the film thickness portion excluding the volume of the hollow portion is preferably 20% to 90%.

[Protein separation and purification method]
The target protein can be separated and purified using the porous membrane having an affinity function according to this embodiment. Hereinafter, the separation and purification method will be described in detail.

  The target recombinant protein can be produced using Escherichia coli, yeast, animal cells and the like as hosts. Alternatively, it can be produced in a cell-free synthesis system. At this time, since impurity proteins are also produced in addition to the target protein, it is necessary to separate and purify them. In order to separate and purify the target protein as it is, a skillful technique is required. Therefore, in order to facilitate separation and purification, the target protein may be tagged with a low molecular weight or GST which is an enzyme. It is preferably produced as a fusion protein in which Among them, a GST fusion protein that expresses a large amount of protein, is easily recovered into a soluble fraction, and can be purified with a small number of processing processes is preferable. The GST fusion protein has a target protein and GST bound to the target protein.

  The recombinant protein produced by adding the tag is then crushed and lysed by cells and cells. The solution to be treated (dissolved solution containing GST fusion protein) containing the dissolved target protein and disrupted cells is filtered by the porous membrane according to the present embodiment, and the target protein can be bound to the affinity ligand in the porous membrane. .

  As described above, when the porous membrane according to the present embodiment is used, it is not necessary to previously separate the cell lysate by centrifugation or a filter, so that the separation and purification process is simplified compared to affinity chromatography using beads as a carrier. The target protein can be obtained from a small amount of sample in a short time.

  After filtration, the impurity protein weakly and non-specifically bound to the affinity ligand of the porous membrane is removed by washing with a buffer solution such as a phosphate buffer.

  Subsequently, the target protein is eluted from the porous membrane using the elution buffer, and the target protein separated and purified can be obtained. As the elution buffer, for example, a solution obtained by adding reduced glutathione to a buffer solution such as TRIS buffer or phosphate buffer can be used.

  When separating and purifying proteins, a flat membrane-like porous membrane can be used while being fixed to a holder. In the case of a hollow fiber-like porous membrane, the porous membrane can be used in the form of a membrane module in which a plurality of porous membranes are bundled and fixed to a plastic housing or the like using an epoxy resin or a urethane resin.

  The driving force for filtration may be pressurization using a pump or air. A flat membrane-like porous membrane set in a holder can be installed in a syringe or the like and manually filtered under pressure. The operating conditions such as pressure and filtration flow rate when filtering the treatment liquid containing protein with a porous membrane are such that if the filtration pressure is too high, the membrane will be easily damaged, and if the filtration flux is too high, the target protein will become an affinity ligand. Since it cannot be captured and it is easy to escape, it can be selected appropriately in consideration of these factors.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1]
(Synthesis of glutathione and an organic compound having a hydrophobic group bonded to glutathione: GSSG compound and GSH compound)
In 10 mL of N, N-dimethylformamide (DMF), 306.3 mg (0.5 mmol) of glutathione (oxidized form), 137.9 mg (0.55 mmol) of 1-bromohexadecane, 103.67 mg (0.75 mmol) of potassium carbonate. The mixture was mixed and refluxed at 50 ° C. for 24 hours under a nitrogen atmosphere. After the reaction, DMF was distilled off under reduced pressure using an evaporator. The residue was washed twice with diethyl ether, then an excess amount of 1M hydrochloric acid was added, and chloroform was further added. The aqueous phase was discarded using a separatory funnel, and the chloroform phase was washed once with 1M hydrochloric acid and once with ultrapure water. The chloroform phase was collected and chloroform was distilled off under reduced pressure using an evaporator, and then the residue was freeze-dried to obtain oxidized glutathione and a compound having a hydrophobic group bonded to oxidized glutathione (GSSG compound). The obtained GSSG compound was reduced with tributylphosphine. Nano-electrospray mass spectrometry (ESI-MS) was performed on the obtained reaction product. As a result, it was confirmed that the reaction product was an organic compound (GSH compound) represented by the chemical formula (1) having glutathione (reduced form) as a ligand and a hexadecyl group as a hydrophobic group bound to glutathione. It was done.

(Manufacture of porous membrane with affinity function)
Dissolve 2.3 g of cellulose diacetate, 115 mg of GSSG compound and 82 mg of meltcaptoethanol (10 times molar equivalent of GSSG compound) in 7.7 g of triethylene glycol, an organic liquid, at 150 ° C. to obtain a uniform polymer solution (manufactured Membrane undiluted solution) was obtained. This polymer solution was applied to a glass plate placed on a heated hot stage, and the glass plate was covered on the applied polymer solution. Thereafter, the polymer solution was solidified and phase-separated by gradually cooling in a room temperature atmosphere to form a polymer thin film as a porous membrane precursor. The obtained polymer thin film was washed with water to remove triethylene glycol remaining in the film, thereby obtaining a thin porous film. When the surface, back surface, and cross section of the obtained porous film were observed using FE-SEM, it was confirmed that pores having a pore diameter of about 500 nm were formed by cellulose acetate. From this result, it was confirmed that the porous membrane has a pore size that allows the impurity protein to permeate while removing cell debris having a size on the order of several μm.

(Confirmation of target protein capture performance)
GSH compound having the above-mentioned ligand is immobilized from an aqueous solution containing TRITC-GST in which glutathione-S-transferase (GST), which is the target protein, is fluorescently labeled with TRITC (Tetramethylrhodamine-5- (and 6) -isothiocynate) Separation and purification of the target protein using the prepared porous membrane was performed according to the following procedures (1) to (5). A porous membrane cut out into a circle having a diameter of 25 mm was set in a holder, and this holder was attached to a cylinder for filtration.
(1) Cleaning of porous membrane: The porous membrane was washed by passing 10 mL of deionized water through the porous membrane.
(2) Equilibration of porous membrane: 10 mL of a phosphate buffer solution having a pH of 7.3 was passed through to condition the porous membrane.
(3) Addition of target protein: 4 mL of a solution (stock solution) containing 43 μg / mL TRITC-GST was passed through the porous membrane. The mass of protein in the stock solution (hereinafter referred to as “stock solution fraction”) and the mass of the protein in the filtrate (hereinafter referred to as “non-adsorbed fraction”) are measured by fluorescence measurement of the concentration of TRITC-GST in the solution ( Excitation wavelength: 544 nm, fluorescence wavelength: 572 nm). The results are shown in FIG. In FIG. 2, “GST” indicates TRITC-GST. The mass in the stock solution fraction of TRITC-GST was 172 μg. On the other hand, the mass in the non-adsorbed fraction was 23 μg. It was possible to capture 87% by mass of the target protein, TRITC-GST, in the porous membrane.
(4) Washing the porous membrane: The porous membrane was washed by passing 10 mL of a phosphate buffer solution having a pH of 7.3 through the porous membrane. The mass of protein in the filtrate at this time (hereinafter referred to as “through fraction”) was 43 μg. Here, TRITC-GST eluted at the time of washing is mainly a component remaining in the holder or the cylinder.
(5) Elution of the target protein: 5 mL of an elution buffer having a pH of 7.3 (mixture containing 50 mM Tris-HCl buffer and 10 mM glutathione) was passed through the porous membrane to elute the target protein. The mass of the protein in the obtained filtrate (hereinafter referred to as “elution fraction”) was 65 μg, and it was confirmed that the target protein, TRITC-GST, could be efficiently captured. From this result, it is clear that the GST fusion protein can also be efficiently captured in the same manner as TRITC-GST.

(Confirmation of nonspecific adsorption of impurity proteins)
In the above procedure (3), except that 2 mL of enhanced green fluorescence protein (His-tag EGFP) having a histidine residue (His-tag) at the N-terminus was passed as an impurity protein at a concentration of 45 μg / mL ( The adsorption performance of His-tag EGFP on the porous membrane was evaluated according to the procedures of 1) to (5). The concentration of EGFP was determined by fluorescence measurement (excitation wavelength: 488 nm, fluorescence wavelength: 510 nm). The results are shown in FIG. The masses of the His-tag EGFP stock solution fraction, the non-adsorbed fraction, the flow-through fraction, and the elution fraction were 90 μg, 50 μg, 22 μg, and 3 μg, respectively. About 80% of the supplied impurity protein, His-tag EGFP, is a non-adsorbed fraction that is not adsorbed to the membrane at all, or a flow-through fraction that is weakly adsorbed to the membrane, and the eluted fraction adsorbed to the membrane is about It was confirmed that it was very low at 3%. From the above, it was confirmed that the porous membrane on which the GSH compound is immobilized selectively captures the GST fusion protein, but there is very little non-specific adsorption by other impurity proteins.

[Example 2]
In the procedure (3), using the porous membrane prepared in Example 1, BRI (FITC-BSA) labeled with TRITC-GST as the target protein and fluorescein isothiocyanate (FITC) as the impurity protein, TRITC-GST was separated and purified according to the procedure of (Separation and purification of protein) of Example 1 except that 2 mL of the mixed solution having a protein concentration of 45 μg / mL was filtered. The concentration of FITC-BSA in the solution was determined by fluorescence measurement (excitation wavelength: 495 nm, fluorescence wavelength: 525 nm). The results are shown in FIG. In FIG. 4, “BSA” indicates FITC-BSA, and “GST” indicates TRITC-GST. The mass of the target protein TRITC-GST in the stock solution fraction, the non-adsorbed fraction, the flow-through fraction, and the elution fraction is 90 μg, 14 μg, 22 μg, and 54 μg, respectively, and BSA coexists as an impurity protein. It was also confirmed that GST protein can be selectively recovered. On the other hand, the masses of the stock solution fraction, non-adsorbed fraction, flow-through fraction, and eluted fraction of FITC-BSA, which is an impurity protein, were 90 μg, 9 μg, 15 μg, and 5 μg, respectively.

  The present invention has industrial applicability in the field of protein separation and purification.

  DESCRIPTION OF SYMBOLS 1 ... Polymer phase, 2 ... Pore, 3 ... Glutathione and organic compound which has hydrophobic group couple | bonded with glutathione (ligand compound), 4 ... Hydrophobic group, 5 ... Ligand (glutathione), 6 ... GST fusion protein, 7 ... porous membrane.

Claims (2)

A polymer, an organic liquid which dissolves the polymer, and having a hydrophobic group attached to the glutathione glutathione to give an organic compound, a porous membrane precursor formed by the polymer solution is phase separation comprises the step ,
Removing the organic liquid from the porous film precursor to form a porous film containing the polymer and the organic compound,
The polymer is cellulose diacetate;
The organic liquid is triethylene glycol;
The hydrophobic group is a hexadecyl group,
A method for producing a porous membrane. A solution containing a glutathione-S-transferase fusion protein is filtered through a porous membrane obtainable by the method according to claim 1, and the glutathione-S-transferase fusion protein is adsorbed to the porous membrane by glutathione-S-transferase fusion protein. A method for separating and purifying an S-transferase fusion protein.

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