Extraction reagents for recombinant proteins

Abstract

To provide an extraction reagent capable of efficiently extracting an Fc receptor in culturing a transformant, which is obtained by genetically transforming a coliform bacillus by using a vector including polynucleotide encoding the receptor, and extracting the receptor from the cultured transformant, and a method for producing the Fc receptor by using the reagent.SOLUTION: The problem is solved by an extraction reagent containing at least nonionic surfactant and halide ion or anionic surfactant.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a reagent that can efficiently extract an Fc receptor from Escherichia coli capable of expressing the receptor, and a method for producing the receptor using the reagent. [Background technology]

[0002] As a method for transforming Escherichia coli with a vector containing a polynucleotide encoding a protein, and culturing the resulting transformant, and extracting the protein from the cultured transformant, conventionally known methods include physical disruption methods such as ultrasonic disruption and French press treatment, and chemical treatment methods using commercially available extraction reagents. However, when attempting to extract the protein industrially using these methods, physical disruption methods have the problem of requiring enormous costs for installing a disruption device such as an ultrasonic disrupter or a French press, and chemical treatment methods have the problem of using a large amount of expensive extraction reagents.

[0003] In addition to the above-mentioned methods, various methods using surfactants are known as methods for extracting the protein, such as a method of extraction using a reagent containing a nonionic surfactant and an anionic surfactant such as a cholic acid (Patent Document 1), a method of extraction using a reagent containing a cationic surfactant (Patent Document 2), and a method of extraction using a reagent containing a nonionic surfactant and a cationic surfactant (Patent Document 3). However, when the protein is a protein whose stability has been improved by amino acid substitution, such as the Fc-binding protein described in Patent Document 4, it is hydrophobic compared to proteins having naturally occurring amino acid sequences, and this has led to the problem that efficient extraction is not possible. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-145172 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-320313 [Patent Document 3] Japanese Patent Application Laid-Open No. 2013-252099 [Patent Document 4] Japanese Patent Publication No. 2020-092689 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a reagent that can efficiently extract an Fc receptor when transforming Escherichia coli with a vector containing a polynucleotide encoding the receptor and culturing the transformant to extract the receptor from the cultured transformant, and a method for producing the receptor using the reagent. [Means for solving the problem]

[0006] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that Fc receptors (hereinafter also referred to as FcRs) can be efficiently extracted from transformants by adding at least a nonionic surfactant and a halide ion or anionic surfactant to the extraction reagent.

[0007] That is, the present invention is as follows: <1> from <7> This includes aspects described in: <1> A reagent for extracting a recombinant protein expressed in Escherichia coli cells capable of expressing the protein, wherein the recombinant protein is FcR, and the reagent contains at least a nonionic surfactant and, as an additive, a halide ion or an anionic surfactant.

[0008] <2> The additive is chloride ion at a final concentration of 0.5 mol / L to 2 mol / L. <1> The reagent described in

[0009] <3> The additive is sodium dodecyl sulfate at a final concentration of 0.05 (w / v)% to 2 (w / v)%. <1> The reagent described in

[0010] <4> Further containing a carbohydrate-degrading enzyme, <1> from <3> The reagent according to any one of the preceding items.

[0011] <5> Further comprising a nuclease, <1> from <4> The reagent according to any one of the preceding items.

[0012] <6> A method for producing an FcR, comprising the steps of culturing Escherichia coli capable of expressing an FcR, and extracting the FcR from the cells of the cultured Escherichia coli using an extraction reagent containing at least a nonionic surfactant and a halide ion or an anionic surfactant.

[0013] <7> The method includes a step of suspending E. coli cells obtained by culturing E. coli capable of expressing FcR in a buffer solution in an amount of buffer 6 to 30 times the amount of the cells, thereby extracting FcR. <6> The method for producing the FcR described above.

[0014] The present invention will be described in detail below.

[0015] The extraction reagent of the present invention is a reagent that can extract the protein expressed in Escherichia coli by transforming the transformant with a vector containing a polynucleotide encoding a recombinant protein, without mechanical disruption, by dissolving the cell wall of the transformant. The extraction reagent is characterized by containing at least a nonionic surfactant and a halide ion or an anionic surfactant.

[0016] The nonionic surfactant contained in the extraction reagent of the present invention can be appropriately selected from among those commonly used as membrane protein solubilizing agents, and examples thereof include Triton X-100 (trade name), Triton X-114 (trade name), Brij 58 (trade name), Brij 35 (trade name), Tween 20 (trade name), Tween 80 (trade name), 1-On-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-dodecyl-β-D-maltopyranoside, n-dodecyl-α-D-maltopyranoside, n-dodecyl-N,N-dimethylamine-N-oxide, isopropyl-β-D-thiogalactoside, sucrose monododecanoate, n-octyl-β-D-glucopyranoside, n-dodecyl-β-D-maltopyranoside, n-tridecyl-β-D-maltopyranoside, etc. The concentration of the nonionic surfactant contained in the extraction reagent of the present invention can be appropriately determined taking into consideration the critical micelle concentration of the surfactant used and the properties and concentrations of impurities in the culture medium, such as cell lysates. For example, when Triton X-100 is used as the nonionic surfactant, the final concentration is preferably set in the range of 0.2 (w / v) % to 2 (w / v) %.

[0017] When halide ions are used as additives in the extraction reagent of the present invention, the additive halide ions can be generated in the solution by adding a halide such as an alkali metal, alkaline earth metal, or ammonia to the extraction reagent of the present invention. For example, when chloride ions are used as halide ions, chlorides such as alkali metals, alkaline earth metals, or ammonia can be added to the extraction reagent to a final concentration preferably in the range of 0.5 mol / L to 2 mol / L, more preferably in the range of 0.8 mol / L to 1.5 mol / L.

[0018] When an anionic surfactant is used as an additive in the extraction reagent of the present invention, it can be appropriately selected from among those commonly used as membrane protein solubilizers. Examples include dodecyl sulfate, cholic acid, deoxycholic acid, glycolic acid, taurocholic acid, taurodeoxycholic acid, and their salts. As with nonionic surfactants, the concentration of the anionic surfactant can be appropriately determined based on the critical micelle concentration of the surfactant used and the properties and concentration of contaminants in the culture medium, such as cell lysates. For example, when using sodium dodecyl sulfate (SDS), a dodecyl sulfate salt, as the anionic surfactant, the final concentration is preferably set in the range of 0.05% (w / v) to 2% (w / v), more preferably in the range of 0.1% (w / v) to 1.5% (w / v), and even more preferably in the range of 0.2% (w / v) to 1.0% (w / v).

[0019] The extraction reagent of the present invention preferably further contains a carbohydrate-degrading enzyme, since this promotes lysis of the transformant (E. coli). The carbohydrate-degrading enzyme that may be contained in the extraction reagent of the present invention can be appropriately selected from among those commonly used as membrane protein solubilizers, such as lysozyme, cellulase, pectinase, and salts thereof. The concentration of the carbohydrate-degrading enzyme can be appropriately determined taking into account the properties and concentrations of impurities in the culture medium, such as cell lysates. For example, when human-derived lysozyme is used as the carbohydrate-degrading enzyme, the final concentration should be set in the range of 0.0001% (w / v) to 0.01% (w / v).

[0020] The extraction reagent of the present invention may contain components other than those described above. In particular, it is preferable to further contain an endonuclease (nuclease) such as Benzonase (Merck & Co.), because this can suppress the increase in viscosity caused by nucleic acids extracted from transformants along with proteins by the extraction reagent. When the extraction reagent of the present invention further contains a nuclease, it is preferable to include magnesium sulfate at a final concentration of about 2 mmol / L as an auxiliary agent.

[0021] In addition to nucleases, components that may be further contained in the extraction reagent of the present invention include buffer components and salts. Examples of buffer components include acetate buffers with concentrations of about 10 mmol / L to 100 mmol / L, citrate-phosphate buffers, phosphate buffers, glycine-NaOH buffers, MES (2-Morpholinoethanesulfonic acid) buffers, ADA (N-(2-Acetamido)iminodiacetic acid) buffers, PIPES (Piperazine-1,4-bis(2-ethanesulfonic acid)) buffers, ACES (N-(2-Acetamido)-2-aminoethanesulfonic acid) buffers, cholamine-HCl buffers, BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid) buffers, TES (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffers, HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic Examples of suitable buffer solutions include tricine (N-[Tris(hydroxymethyl)methyl]glycine) buffer, acetamidoglycine buffer, Tricine (N-[Tris(hydroxymethyl)methyl]glycine) buffer, glycinamide buffer, and Bicine (N,N-Bis(2-hydroxyethyl)glycine) buffer. An example of a preferred buffer solution is phosphate buffer with a concentration of 20 mmol / L to 50 mmol / L and a pH of 6.0 to 8.0. Examples of salts include sulfates, phosphates, acetates, citrates, carbonates, and the like with a final concentration of 10 mmol / L to 1000 mmol / L.

[0022] When a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding a recombinant protein is used to extract the protein from the transformant using the extraction reagent of the present invention, the efficiency of protein extraction is improved by performing the extraction procedure under pH conditions (specifically, pH 5.0 to 9.0) at which the protein is not inactivated and at which the additionally added enzymes (carbohydrate-degrading enzymes and nucleases) are active. Setting the pH in the range of pH 5.5 to 6.5 is particularly preferred.

[0023] After extracting a protein from a transformant using the extraction reagent of the present invention, a purification procedure is performed to remove extraction residues (such as cell debris) using a method known in the art, thereby obtaining the protein with a high purity. One example of the purification procedure is separation and purification using liquid chromatography. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and affinity chromatography. By performing the purification procedure using a combination of these chromatographic methods, the protein can be produced with a high purity.

[0024] When performing purification using liquid chromatography, if the protein solution obtained in the extraction step has high turbidity (i.e., low clarity), problems such as poor protein separation ability and reduced column performance reproducibility can occur. Therefore, the higher the clarity of the protein solution obtained in the extraction step, the better. Therefore, it is recommended to first increase the clarity of the protein solution by centrifugation or filtration before performing purification using chromatography. Methods for increasing the clarity of a protein solution by centrifugation include batch centrifugation using dedicated bottles and continuous centrifugation such as cylindrical and plate-type centrifugation. Methods for increasing the clarity of a protein solution by filtration include methods using MF membranes (microfiltration membranes) and UF membranes (ultrafiltration membranes), and pressure filtration using a filter aid. There are no particular limitations on the type of membrane used for filtration (e.g., material, pore size, molecular weight cutoff, etc.), as long as it can efficiently remove insoluble matter.

[0025] The method for analyzing the protein obtained by the production method of the present invention is not particularly limited as long as it can be stably and efficiently quantified from the culture medium, extract, etc., and examples include ELISA (enzyme-linked immunosorbent assay) and Western blotting.

[0026] The FcR protein extracted using the extraction reagent of the present invention is not particularly limited, as long as it can be expressed in the cells of a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding the FcR. Known FcRs include the Fcγ receptor (FcγR), which is a receptor for IgG (immunoglobulin G), the Fcε receptor that binds to the Fc region of IgE, and the Fcα receptor that binds to the Fc region of IgA, and all of these can be extracted with the extraction reagent of the present invention. When the FcR extracted with the extraction reagent of the present invention is FcγR, examples include FcγRIa, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. Of these, FcγRIIIa is preferred as the FcR extracted with the extraction reagent of the present invention.

[0027] Examples of FcγRIIIa extracted using the extraction reagent of the present invention include: a polypeptide comprising at least the extracellular region of human native FcγRIIIa (the amino acid residues from glycine at position 17 to glycine at position 193 of the amino acid sequence set forth in SEQ ID NO: 1 [UniProt No. P08637]); a polypeptide comprising at least the amino acid residues of the extracellular domain, wherein one or more amino acid residues between the 17th and 193rd amino acid residues are deleted, substituted with other amino acid residues, or added; Among these, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2 is preferred as the FcγRIIIa. The amino acid residues from glycine 17 to glycine 193 in the amino acid sequence and wherein the 17th to 193rd amino acid residues have the amino acid substitutions shown in (1) to (36) below: (1) Glutamic acid at position 21 of SEQ ID NO: 1 (position 5 of SEQ ID NO: 2) is replaced with glycine (2) Leucine at position 23 of SEQ ID NO: 1 (position 7 of SEQ ID NO: 2) is replaced with methionine (3) Valine at position 27 of SEQ ID NO: 1 (position 11 of SEQ ID NO: 2) is replaced with glutamic acid (4) Phenylalanine at position 29 of SEQ ID NO: 1 (position 13 of SEQ ID NO: 2) is substituted with isoleucine (5) Substitution of glutamine at position 33 of SEQ ID NO: 1 (position 17 of SEQ ID NO: 2) with proline (6) Tyrosine at position 35 of SEQ ID NO: 1 (position 19 of SEQ ID NO: 2) is substituted with asparagine (7) Lysine at position 40 of SEQ ID NO: 1 (position 24 of SEQ ID NO: 2) is replaced with glutamine (8) Glutamine at position 48 of SEQ ID NO: 1 (position 32 of SEQ ID NO: 2) is replaced with arginine (9) Tyrosine at position 51 of SEQ ID NO: 1 (position 35 of SEQ ID NO: 2) is substituted with histidine (10) Glutamic acid at position 54 of SEQ ID NO: 1 (position 38 of SEQ ID NO: 2) is replaced with aspartic acid (11) Asparagine at position 56 of SEQ ID NO: 1 (position 40 of SEQ ID NO: 2) is substituted with aspartic acid (12) Serine at position 65 of SEQ ID NO: 1 (position 49 of SEQ ID NO: 2) is substituted with arginine (13) Serine at position 68 of SEQ ID NO: 1 (position 52 of SEQ ID NO: 2) is substituted with proline (14) Tyrosine at position 74 of SEQ ID NO: 1 (position 58 of SEQ ID NO: 2) is substituted with phenylalanine (15) Phenylalanine at position 75 of SEQ ID NO: 1 (position 59 of SEQ ID NO: 2) is substituted with isoleucine (16) Alanine at position 78 of SEQ ID NO: 1 (position 62 of SEQ ID NO: 2) is substituted with serine (17) Threonine at position 80 of SEQ ID NO: 1 (position 64 of SEQ ID NO: 2) is substituted with serine (18) Asparagine at position 92 of SEQ ID NO: 1 (position 76 of SEQ ID NO: 2) is substituted with serine (19) Valine at position 117 of SEQ ID NO: 1 (position 101 of SEQ ID NO: 2) is substituted with glutamic acid (20) Lysine at position 119 of SEQ ID NO: 1 (position 103 of SEQ ID NO: 2) is substituted with valine (21) Glutamic acid at position 121 of SEQ ID NO: 1 (position 105 of SEQ ID NO: 2) is replaced with glycine (22) Aspartic acid at position 122 of SEQ ID NO: 1 (position 106 of SEQ ID NO: 2) is substituted with glutamic acid (23) Lysine at position 132 of SEQ ID NO: 1 (position 117 of SEQ ID NO: 2) is substituted with arginine (24) Threonine at position 140 of SEQ ID NO: 1 (position 124 of SEQ ID NO: 2) is replaced with methionine (25) Tyrosine at position 141 of SEQ ID NO: 1 (position 125 of SEQ ID NO: 2) is substituted with phenylalanine. (26) Glycine at position 147 of SEQ ID NO: 1 (position 131 of SEQ ID NO: 2) is substituted with valine (27) Tyrosine at position 158 of SEQ ID NO: 1 (position 142 of SEQ ID NO: 2) is replaced with valine (28) Lysine at position 165 of SEQ ID NO: 1 (position 149 of SEQ ID NO: 2) is substituted with glutamic acid (29) Phenylalanine at position 171 of SEQ ID NO: 1 (position 155 of SEQ ID NO: 2) is substituted with serine (30) Phenylalanine at position 176 of SEQ ID NO: 1 (position 160 of SEQ ID NO: 2) is substituted with isoleucine (31) Serine at position 178 of SEQ ID NO: 1 (position 162 of SEQ ID NO: 2) is substituted with arginine (32) Asparagine at position 180 of SEQ ID NO: 1 (position 164 of SEQ ID NO: 2) is substituted with lysine (33) Glutamic acid at position 184 of SEQ ID NO: 1 (position 168 of SEQ ID NO: 2) is replaced with glycine (34) Threonine at position 185 of SEQ ID NO: 1 (position 169 of SEQ ID NO: 2) is substituted with alanine (35) Asparagine at position 187 of SEQ ID NO: 1 (position 171 of SEQ ID NO: 2) is substituted with aspartic acid (36) Isoleucine at position 190 of SEQ ID NO: 1 (position 174 of SEQ ID NO: 2) is substituted with valine. [Effects of the Invention]

[0028] The extraction reagent of the present invention is characterized by containing at least a nonionic surfactant and a halide ion or an anionic surfactant, and can efficiently extract an Fc receptor expressed in a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding the receptor.

[0029] The extraction reagent of the present invention is particularly effective for extracting proteins that are more hydrophobic than proteins with naturally occurring amino acid sequences, such as Fc receptors whose stability has been improved by amino acid substitutions in the amino acid sequence of naturally occurring proteins.

[0030] Furthermore, the method for extracting the receptor using the extraction reagent of the present invention can extract the receptor more cheaply and efficiently than methods that involve physical disruption or methods that use commercially available extraction reagents, and therefore can be said to be useful in the industrial production of Fc receptors. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows the results of examining additives to be contained in extraction reagents. [Figure 2] FIG. 1 shows the results of examining the concentration of sodium dodecyl sulfate contained in an extraction reagent. [Figure 3] FIG. 10 shows the results of examining the amount of extraction reagent. [Example]

[0032] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0033] Example 1: Examination of additives (1) Recombinant E. coli was obtained by transforming Escherichia coli W3110 with an expression vector containing a polynucleotide (SEQ ID NO: 3) encoding an Fc receptor (hereinafter also referred to as FcR) consisting of the amino acid sequence set forth in SEQ ID NO: 2. The recombinant E. coli was cultured with reference to the method described in JP 2013-085531 A, and E. coli cells (wet cells) were obtained from the resulting culture medium by centrifugation.

[0034] (2) The wet cells obtained in (1) were suspended in 20 mmol / L phosphate buffer (pH 6.0) containing 1 mmol / L EDTA in an amount three times the weight of the wet cells, and the suspension was stirred at room temperature (20 to 25°C) until homogenous.

[0035] (3) The bacterial cell suspension obtained in (2) was dispensed into a 96-well microwell plate in 500 μL aliquots, and then Benzonase (Merck), a nuclease, was added to each well to a final concentration of 2500 units / L. Magnesium sulfate was added as an auxiliary agent to a final concentration of 2 mmol / L, and the mixture was stirred at room temperature.

[0036] (4) After adding one of the additives (a) to (q) below (all concentrations are final concentrations), lysozyme, a carbohydrate-degrading enzyme, was added to a final concentration of 0.005 (w / v)%, and the mixture was further stirred at room temperature.

[0037] (a) Sodium chloride 1 mol / L (b) β-mercaptoethanol 1(v / v)% (c) β-mercaptoethanol 5(v / v)% (d) Sodium sulfate 0.1 mol / L (e) Sodium sulfate 1 mol / L (f) Urea 0.1mol / L (g) Urea 1mol / L (h) Arginine hydrochloride 0.1 mol / L (i) Arginine hydrochloride 1 mol / L (j) Guanidine hydrochloride 0.1 mol / L (k) Guanidine hydrochloride 1 mol / L (l)Tween 20 (product name) 0.1(v / v)% (m)Tween 20 (product name) 1(v / v)% (n) Sodium deoxycholate 0.001(w / v)% (o) Sodium deoxycholate 0.01(w / v)% (p) Sodium dodecyl sulfate 0.1(w / v)% (q) Sodium dodecyl sulfate 1(w / v)% (5) An aqueous solution of Triton X-100 (trade name), a nonionic surfactant, was added to a final concentration of 0.5 (w / v)%, and the mixture was stirred at room temperature to extract FcR.

[0038] (6) After overnight extraction, the extract from (5) was centrifuged (3000 rpm, 20 minutes, twice) to obtain the supernatant (cell-free extract).

[0039] (7) The amount of FcR extracted from the cell-free extract obtained in (6) was measured by the ELISA method described below. (7-1) A gamma globulin preparation (manufactured by Chemo-Sero-Therapeutic Research Institute), which is a human-derived antibody, was immobilized in the wells of a 96-well microplate at a concentration of 10 μg / well (at 4° C. for 18 hours or more). (7-2) After immobilization, the membrane was washed with a washing buffer (20 mmol / L Tris-HCl buffer (pH 7.5) containing 0.05 (w / v)% Tween 20 (trade name) and 150 mmol / L sodium chloride) and then blocked with 1 (w / v)% BSA (Sigma-Aldrich) at 4°C overnight. (7-3) After washing with the washing buffer, the prepared cell-free extract was appropriately diluted with 50 mmol / L Tris-HCl buffer (pH 7.4) and reacted with the immobilized gamma globulin at 30°C for 1 hour. (7-4) After the reaction was completed, the plate was washed again with the washing buffer, and an anti-hFcγRIII antibody reagent (R&D SYSTEMS, MAB2546) was added, followed by reaction at 30° C. for 1 hour. (7-5) After the reaction was completed, the plate was washed again with the washing buffer. Horse radish peroxidase (HRP)-labeled anti-mouse antibody reagent (BETHYL, A90-216P) was added and the mixture was allowed to react at 30°C for 1 hour. (7-6) After incubation at 30°C for 1 hour, the mixture was washed with washing buffer. TMB Peroxidase Substrate (KPL) was added, and after incubation for a desired time, a 1 mol / L aqueous phosphoric acid solution was added to terminate the reaction, and the absorbance at 450 nm was measured.

[0040] Example 2: Examination of sodium dodecyl sulfate concentration (1) The same procedures as in Example 1 (1) and (2) were carried out to obtain a bacterial cell suspension.

[0041] (2) To the bacterial cell suspension obtained in (1), Benzonase (Merck), a nuclease, was added to a final concentration of 2500 units / L, and magnesium sulfate was added as an auxiliary agent to a final concentration of 2 mmol / L, followed by stirring at room temperature (20°C to 25°C).

[0042] (3) The bacterial cell suspension obtained in (2) was dispensed into five beakers at 20 mL each, and sodium dodecyl sulfate was added to each beaker to a final concentration of 0, 0.1, 0.2, 1.0, or 2.0 w / v%. Lysozyme was then added to a final concentration of 0.005 (w / v)%, and the mixture was stirred at room temperature (20 to 25°C).

[0043] (4) An aqueous solution of Triton X-100 (trade name), a nonionic surfactant, was added to a final concentration of 0.5 (w / v)%, and the mixture was stirred at room temperature (20°C to 25°C) to extract FcR.

[0044] (5) After overnight extraction, the extract was centrifuged (9000 rpm, 20 minutes) to obtain the supernatant (cell-free extract).

[0045] (6) The amount of extracted FcR was measured by the method described in Example 1(7).

[0046] The results are shown in Figure 2. The addition of sodium dodecyl sulfate at final concentrations of 0.1 w / v% to 2.0 w / v% improved the amount of FcR extracted compared to when no sodium dodecyl sulfate was added. In particular, when sodium dodecyl sulfate was added at a final concentration of 1 w / v%, the amount of extraction was significantly improved compared to when no sodium dodecyl sulfate was added.

[0047] Comparative Example 1 The same procedure as in Example 1 (4) was carried out except that no additive was added.

[0048] Comparative Example 2 (1) The bacterial cell suspension obtained in Example 1(2) was extracted using a commercially available extraction reagent (BugBuster, manufactured by Merck) according to the standard protocol attached to the reagent.

[0049] (2) The extract was centrifuged to obtain the supernatant (cell-free extract).

[0050] (3) The amount of extracted FcR was measured by the method described in Example 1(7).

[0051] The results of comparing the FcR extraction amounts in Example 1 and Comparative Examples 1 and 2 are shown in Figure 1, and the evaluation results of the extraction amount and separability depending on the additive used in Example 1 are shown in Table 1. In Table 1, the extraction amount was evaluated as "○" if the extraction amount was greater than that without additive (Comparative Example 1), "△" if the extraction amount was less than that without additive but greater than that of the commercially available extraction reagent (Comparative Example 2), and "×" if the extraction amount was less than that of the commercially available extraction reagent (Comparative Example 2). Separability was evaluated as "○" if the interface between the supernatant and precipitate was clearly separated by centrifugation, and the supernatant alone could be easily recovered without any sediment mixing, and "×" otherwise.

[0052] [Table 1]

[0053] Table 1 shows that when sodium chloride was added to a final concentration of 1 mol / L (Figure 1(a)), and sodium dodecyl sulfate was added to a final concentration of 0.1 (w / v)% (Figure 1(p)) or 1 (w / v)% (Figure 1(q)), the extraction yield was superior to that of the extraction reagent without additives (Comparative Example 1) and the commercially available extraction reagent (Comparative Example 2), and the separation of the extract was also high. This shows that FcR expressed in E. coli can be efficiently extracted using an extraction reagent in which a halide ion or anionic surfactant is added as an additive to a nonionic surfactant.

[0054] Example 3: Examination of the amount of extraction reagent (1) The same procedure as in Example 1(1) was carried out to obtain Escherichia coli cells (wet cells).

[0055] (2) The wet bacterial cells obtained in (1) were suspended in 20 mmol / L phosphate buffer (pH 6.0) containing 1 mmol / L EDTA in an amount 3, 4, 6, 10, 15, 20, or 30 times the weight of the wet bacterial cells, and stirred at room temperature (20°C to 25°C) until homogenous.

[0056] (3) To the bacterial cell suspension obtained in (2), Benzonase (Merck), a nuclease, was added to a final concentration of 2500 units / L, and magnesium sulfate was added as an auxiliary agent to a final concentration of 2 mmol / L, followed by stirring at room temperature (20°C to 25°C).

[0057] (4) Sodium dodecyl sulfate was added to the bacterial cell suspension obtained in (3) to a final concentration of 0.2 (w / v)% and stirred at room temperature. Lysozyme, a carbohydrate-degrading enzyme, was then added to a final concentration of 0.005 (w / v)% and stirred at room temperature.

[0058] (5) The same procedures as in Example 2(4) to (6) were carried out to measure the amount of FcR. The results are shown in Figure 3. When the bacteria were suspended in an extraction reagent six times or more their wet bacterial weight, the amount of extraction was more than doubled compared to when three times the amount of extraction reagent was used. FcR has a lower solubility than common proteins such as protein A and bovine serum albumin (BSA), and using six times or more of the extraction reagent is thought to have prevented insolubilization, thereby significantly improving the amount of extraction. [Industrial Applicability]

[0059] The present invention enables efficient extraction of an Fc receptor expressed in Escherichia coli cells, and therefore allows efficient production of the Fc receptor, which comprises the steps of culturing a transformant obtained by transforming Escherichia coli with a vector containing a polynucleotide encoding the receptor, and extracting the receptor from the cultured transformant.

Claims

1. A reagent for extracting a recombinant protein expressed in Escherichia coli cells capable of expressing the protein, comprising: the recombinant protein is an Fc receptor; The reagent comprises at least Triton X-100 at a final concentration of 0.2 (w / v)% to 2 (w / v)% and sodium dodecyl sulfate at a final concentration of 0.05 (w / v)% to 2 (w / v)%.

2. The reagent of claim 1 , further comprising a glycolytic enzyme.

3. A method for producing an Fc receptor, comprising the steps of culturing Escherichia coli capable of expressing an Fc receptor, and extracting the receptor from the cells of the cultured Escherichia coli using the extraction reagent described in claim 1.

4. The method according to claim 3, comprising the step of suspending Escherichia coli cells obtained by culturing Escherichia coli capable of expressing an Fc receptor in a buffer solution in an amount 6 to 30 times the weight of the cells, thereby extracting the Fc receptor.

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