Method for producing insoluble recombinant protein aggregates

Abstract

The object of this invention is to provide a method for efficiently isolating the insoluble form of a recombinant protein from recombinant cells that express a target recombinant protein intracellularly in an insoluble form. This invention provides a method for manufacturing an insoluble recombinant protein aggregate, which is a method for isolating the insoluble form of a recombinant protein from recombinant cells that express a recombinant protein intracellularly in an insoluble form, wherein the recombinant cells are ruptured, and the insoluble form of the recombinant protein is agglomerated and then separated.

Description

[0001] This application is a divisional application, which targets the Chinese national application number 201780048398.8, the international application number PCT / JP2017 / 029033, the application date of which was August 10, 2017, the entry date into China of which was February 1, 2019, and the invention title of which is "Method for manufacturing insoluble recombinant protein condensate". Technical Field

[0002] The present invention relates to a method for producing an insoluble recombinant protein condensate by means of isolating an insoluble form of the recombinant protein from recombinant cells expressing an insoluble recombinant protein, and to the insoluble recombinant protein condensate obtained by means of the method. Background Technology

[0003] The use of recombinant host cells has enabled the industrial-scale production of target proteins. Many methods for isolating and purifying recombinant proteins produced using recombinant cells have also been reported.

[0004] When recombinant proteins are densely generated as insoluble particles within recombinant cells, these insoluble particles can be separated with high yield and high purity by centrifugation of a suspension containing components such as proteins derived from the host cell. For example, a method for separating the target protein from insoluble recombinant cells that have been soluble using metal hydroxides such as sodium hydroxide has been reported (Patent Document 1), etc.

[0005] On the other hand, when recombinant proteins are in a soluble state within recombinant cells, or even if they are insoluble but not as dense insoluble particles and difficult to separate by centrifugation, purification methods have been reported, for example, as follows. Specifically, methods have been reported that involve hydrolyzing host cell-derived proteins using organic acids such as formic acid or propionic acid, removing host cell-derived insoluble substances by centrifugation, recovering the target recombinant protein in an undenatured state, and purifying it using methods such as chromatography (Patent Document 2), etc. In this report, even with the addition of the organic acid, the target protein remains undenatured and does not aggregate.

[0006] Within recombinant cells, the target recombinant protein is not necessarily generated in the form of insoluble particles. It is known that the formation of insoluble particles varies considerably depending on the properties of the target recombinant protein itself, as well as various parameters of the culture process such as the composition of the culture medium, culture temperature, and formation rate. Therefore, research has been conducted on modifying recombinant proteins or developing efficient production methods to generate large, easily centrifugally separated particles.

[0007] On the other hand, if there were a method that could easily separate fine insoluble substances or particles that are difficult to separate by centrifugation or that take a very long time to separate by centrifugation, it would be extremely beneficial in industry, but such a method has not yet been known.

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: Japanese Patent Publication No. 2013-523665

[0011] Patent Document 2: Japanese Patent Publication No. 2004-503204 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] The object of the present invention is to provide a method for efficiently isolating the insoluble form of a recombinant protein from recombinant cells that express a target recombinant protein in intracellular form, and a method for manufacturing recombinant protein conglomerates using the separation method.

[0014] Methods for solving problems

[0015] The inventors conducted in-depth research on a method for easily separating insoluble bodies or insoluble particles of fine recombinant proteins that are difficult to separate by centrifugation or that take a very long time to centrifuge. As a result, they discovered that by efficiently agglomerating the insoluble bodies or insoluble particles to make them larger, recombinant proteins can be easily separated, thus completing this invention.

[0016] That is, the present invention relates to, for example, the following inventions.

[0017] [1] A method for manufacturing a recombinant protein condensate, comprising: separating the insoluble form of the recombinant protein from recombinant cells expressing the recombinant protein intracellularly in the form of a condensate to manufacture the recombinant protein condensate, wherein: After the recombinant cells are broken down, the insoluble bodies of the recombinant proteins are agglutinated, and the resulting agglutinates are separated.

[0018] [2] The method for manufacturing the recombinant protein condensate as described in [1] includes: separating the recombinant protein condensate by centrifugation at a force of less than 10,000 g.

[0019] [3] A method for manufacturing a recombinant protein condensate as described in [1] or [2], comprising: separating the recombinant protein condensate using a centrifuge selected from the group consisting of a plate centrifuge, a basket centrifuge, and a decanter centrifuge.

[0020] [4] The method for manufacturing the recombinant protein condensate as described in [1] includes: separating the recombinant protein condensate by natural sedimentation or filtration.

[0021] [5] A method for manufacturing a recombinant protein aggregate as described in any one of [1] to [4], wherein the aggregation of the insoluble form of the recombinant protein is carried out by adding one or more selected from the group consisting of metal salts, acids and anionic agglutinants.

[0022] [6] A method for manufacturing a recombinant protein condensate, comprising the steps (A) to (C) below.

[0023] (A) The step of lysing recombinant cells expressing the target recombinant protein in intracellular form as insoluble bodies to obtain a lysed suspension containing the recombinant protein as insoluble bodies.

[0024] (B) Adding one or more ingredients selected from the group consisting of metal salts, acids, and anionic coagulants to the broken suspension obtained in step (A) to cause the insoluble parts of the recombinant protein to aggregate, thus obtaining the recombinant protein aggregate.

[0025] (C) The step of separating the aggregates obtained in step (B) from the suspension.

[0026] [7] The method for manufacturing recombinant protein condensates as described in [6] further includes heating in step (B) above.

[0027] [8] The method for manufacturing recombinant protein condensates as described in [7] further includes stirring in step (B) above.

[0028] [9] A method for manufacturing a recombinant protein condensate as described in any one of [5] to [8], wherein the metal salt is selected from the group consisting of alkaline earth metal salts and earth metal salts.

[0029]

[10] The method for manufacturing recombinant protein condensates as described in [9], wherein the metal salt is selected from the group consisting of alkaline earth metal halides, alkaline earth metal nitrates, alkaline earth metal sulfates, earth metal halides, earth metal nitrates and earth metal sulfates.

[0030]

[11] A method for manufacturing a recombinant protein condensate as described in any one of [5] to

[10] , wherein the acid is an oxyacid.

[0031]

[12] The method for manufacturing recombinant protein condensate as described in

[11] , wherein the oxyacid is selected from the group consisting of acetic acid, sulfuric acid and citric acid.

[0032]

[13] A method for manufacturing a recombinant protein coagulant as described in any one of [5] to

[12] , wherein the anionic coagulant is an anionic coagulant selected from the group consisting of polyacrylate, anionic polyacrylamide and acrylamide-acrylate copolymer.

[0033]

[14] The method for manufacturing the recombinant protein condensate as described in any one of [1] to

[13] , wherein the disruption of the recombinant cells is mechanical disruption.

[0034]

[15] A method for manufacturing a recombinant protein condensate as described in any one of [1] and [6] to

[14] , wherein the separation of the recombinant protein condensate is performed by filtration.

[0035]

[16] A method for manufacturing a recombinant protein condensate as described in any one of [1] to

[15] , wherein the recombinant cell is a recombinant cell transformed with a host selected from the group consisting of bacteria, yeast, filamentous fungi, insect cells, plant cells and animal cells.

[0036]

[17] A method for manufacturing a recombinant protein condensate as described in any one of [1] to

[16] , wherein the recombinant protein is a structural protein.

[0037]

[18] The method for manufacturing recombinant protein condensates as described in

[17] , wherein the structural protein is a protein sourced from the group consisting of keratin, collagen, elastin, arthropod elastin, silk, and spider silk.

[0038]

[19] The method for manufacturing recombinant protein condensates according to any one of [1] to

[18] , wherein the particle size of the recombinant protein condensates obtained by the inductive zone method is 4 μm or more and 50 μm or less.

[0039]

[20] A recombinant protein condensate, which is a recombinant protein condensate obtained by the manufacturing method of any one of [1] to

[18] , wherein the particle size measured by the inductive zone method is 4 μm or more and 50 μm or less.

[0040]

[21] A method for isolating a recombinant protein, comprising: isolating the recombinant protein in its insoluble form from recombinant cells expressing the recombinant protein intracellularly, wherein: After the recombinant cells are broken down, the insoluble bodies of the recombinant proteins are agglutinated, and the resulting agglutinates are separated.

[0041]

[22] The method for separating recombinant proteins as described in

[21] includes: separating the recombinant protein condensate with a centrifugal force of less than 10000×g.

[0042]

[23] The method for separating recombinant proteins as described in

[21] or

[22] includes: separating the recombinant protein aggregates using a centrifuge selected from the group consisting of a plate centrifuge, a basket centrifuge, and a decanter centrifuge.

[0043]

[24] The method for separating recombinant proteins as described in

[21] includes separating the recombinant protein aggregates by natural sedimentation or filtration.

[0044]

[25] The method for separating recombinant proteins as described in any one of

[21] to

[24] , wherein the agglutination of the insoluble form of the recombinant protein is carried out by adding one or more selected from the group consisting of metal salts, acids and anionic agglutinants.

[0045]

[26] A method for isolating a recombinant protein, comprising the steps (A) to (C) below.

[0046] (A) The step of lysing recombinant cells expressing the target recombinant protein in intracellular form as insoluble bodies to obtain a lysed suspension containing the recombinant protein as insoluble bodies.

[0047] (B) Adding one or more ingredients selected from the group consisting of metal salts, acids, and anionic coagulants to the broken suspension obtained in step (A) to cause the insoluble parts of the recombinant protein to aggregate, thus obtaining the recombinant protein aggregate.

[0048] (C) The step of separating the aggregates obtained in step (B) from the suspension.

[0049]

[27] The method for isolating recombinant proteins as described in

[26] , wherein step (B) further includes heating.

[0050]

[28] The method for isolating recombinant proteins as described in

[27] , wherein step (B) further includes stirring.

[0051]

[29] The method for isolating recombinant proteins as described in any one of

[25] to

[28] , wherein the metal salt is selected from the group consisting of alkaline earth metal salts and earth metal salts.

[0052]

[30] The method for separating recombinant proteins as described in

[29] , wherein the metal salt is selected from the group consisting of alkaline earth metal halides, alkaline earth metal nitrates, alkaline earth metal sulfates, earth metal halides, earth metal nitrates and earth metal sulfates.

[0053]

[31] The method for isolating recombinant proteins as described in any one of

[25] to

[30] , wherein the acid is an oxyacid.

[0054]

[32] The method for separating recombinant proteins as described in

[31] , wherein the oxyacid is selected from the group consisting of acetic acid, sulfuric acid and citric acid.

[0055]

[33] The method for separating recombinant proteins as described in any one of

[25] to

[32] , wherein the anionic coagulant is an anionic coagulant selected from the group consisting of polyacrylate, anionic polyacrylamide and acrylamide-acrylate copolymer.

[0056]

[34] The method for separating recombinant proteins as described in any one of

[21] to

[33] , wherein the disruption of the recombinant cells is mechanical disruption.

[0057]

[35] The method for separating recombinant proteins as described in any one of

[21] and

[26] to

[34] , wherein the separation of the recombinant protein condensate is performed by filtration.

[0058]

[36] The method for isolating recombinant proteins as described in any one of

[21] to

[35] , wherein the recombinant cells are recombinant cells transformed with a host selected from the group consisting of bacteria, yeast, filamentous fungi, insect cells, plant cells and animal cells.

[0059]

[37] The method for isolating the recombinant protein as described in any one of

[21] to

[36] , wherein the recombinant protein is a structural protein.

[0060]

[38] The method for isolating recombinant proteins as described in

[37] , wherein the structural protein is a protein sourced from the group consisting of keratin, collagen, elastin, arthropod elastin, silk and spider silk.

[0061]

[39] A method for manufacturing a recombinant protein spheroid, wherein the recombinant protein spheroid is manufactured using the separation method described in any one of [1 to 38], wherein the particle size of the recombinant protein spheroid obtained by the separation method, as determined by the inductive zone method, is 4 μm or more and 50 μm or less.

[0062] Invention Effects

[0063] The method for manufacturing recombinant protein flocculations according to the present invention enables the insoluble particles to aggregate to a large size, thus allowing for the efficient separation of the insoluble portions of the recombinant protein from recombinant cells expressing the target recombinant protein in intracellular form using methods such as natural sedimentation, centrifugation, or filtration. Furthermore, it enables the easy separation of insoluble portions or particles of recombinant proteins that have previously been difficult to separate using centrifugation, filtration, or similar methods, and also improves the purity of the separated recombinant protein. Such unexpected effects are achieved according to the present invention. Attached Figure Description

[0064] Figure 1 This is a photograph showing the results of a study on the aggregation effect of insoluble bodies resulting from the addition of metal salts in Example 1.

[0065] Figure 2 This is a photograph showing the results of a study on the aggregation effect of insoluble bodies resulting from the addition of metal salts in Example 2.

[0066] Figure 3 This is a photograph showing the results of an investigation into the aggregation effect of insoluble proteins with different hydrophilic indices resulting from the addition of metal salts, as described in Example 3.

[0067] Figure 4 This is a photograph showing the results of an investigation into the aggregation effect of insoluble proteins with different hydrophilic indices resulting from the addition of metal salts, as described in Example 4.

[0068] Figure 5 These are photographs illustrating the results of analysis using polyacrylamide gel electrophoresis (SDS-PAGE) in Example 4, related to the increased purity of the target recombinant protein produced by metal salt addition agglutination. Photograph A is obtained after electrophoresis using Oriole (trademark) fluorescent gel staining agent (manufactured by Bio-Rad) capable of staining all proteins. Photograph B is obtained after electrophoresis using InVision (trademark) His-tagged intragel staining reagent (manufactured by Thermo Fisher Scientific) that reacts with the His-tagged region of PRT410.

[0069] Figure 6 This is a photograph showing the results of a study on the aggregation effect of acid addition on insoluble bodies in Example 5.

[0070] Figure 7 This is a photograph showing the results of a study on the agglomeration effect of acid addition on insoluble bodies after cleaning, as described in Example 5.

[0071] Figure 8 This is a photograph showing the results of an investigation into the aggregation effect of insoluble proteins with different hydrophilic indices on the basis of acid addition, as described in Example 6.

[0072] Figure 9 This is a photograph showing the results of SDS-PAGE analysis of Example 6, related to the increased purity of the target recombinant protein produced by acid-added agglutination.

[0073] Figure 10This is a photograph showing the results of a study on the agglomeration effect of adding a coagulant on insoluble bodies, as described in Example 8.

[0074] Figure 11 This is a photograph showing the results of SDS-PAGE analysis of Example 9, related to the increased purity of the target recombinant protein produced by agglutination based on the addition of an anionic agglutinant.

[0075] Figure 12 This is a graph showing the frequency distribution and cumulative distribution of particle size in Example 10 for confirming the agglomeration effect.

[0076] Figure 13 This is a photograph showing the results of an analysis using SDS-PAGE related to the decomposition of heat-induced proteins in Example 12.

[0077] Figure 14 This is a photograph showing the results of an analysis using SDS-PAGE of Example 12, related to the increased purity of the target recombinant protein produced based on heating.

[0078] Figure 15 This is a graph showing the frequency and cumulative distribution of particle size of samples C, X, 1, 2 and 3 in Example 13, used to confirm the agglomeration effect caused by continuous heating.

[0079] Figure 16 This is a graph showing the frequency and cumulative distribution of particle size of samples 4, 5, 6, 7 and 8 in Example 13, used to confirm the agglomeration effect caused by continuous heating. Detailed Implementation

[0080] The specific embodiments of the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[0081] One embodiment of the method for manufacturing a recombinant protein coagulant involves separating the insoluble forms of the recombinant protein from recombinant cells expressing the recombinant protein intracellularly in the form of a coagulant. The method is characterized by comprising: cleaving the recombinant cells, agglutinating the insoluble forms of the recombinant protein, and then separating them. In this manufacturing method, the agglutination of the insoluble forms of the recombinant protein is preferably carried out by adding one or more agents selected from the group consisting of metal salts, acids, and anionic agglutinants.

[0082] Another embodiment of the method for manufacturing a recombinant protein condensate is characterized by including the following steps (A) to (C): (A) The step of lysing recombinant cells that express the target recombinant protein in the form of insoluble bodies to obtain a lysed suspension containing the recombinant protein in the form of insoluble bodies. (B) Add one or more of the group consisting of metal salts, acids and anionic coagulants to the broken suspension obtained in step (A) to cause the insoluble parts of the recombinant protein to aggregate, thereby obtaining a recombinant protein aggregate. (C) The step of separating the aggregates obtained in step (B) from the suspension.

[0083] (Recombinant protein)

[0084] The insoluble recombinant protein (sometimes referred to as the "target protein" in this specification) isolated using the method for manufacturing recombinant protein condensates according to this embodiment is expressed in the following recombinant cells in insoluble form. Examples of recombinant proteins include any insoluble protein suitable for industrial-scale manufacturing, such as proteins suitable for industrial use, proteins suitable for medical use, and structural proteins. Specific examples of proteins suitable for industrial or medical use include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane and transport proteins, antigens used in vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, allergens, full-length antibodies and antibody fragments, and their derivatives. Specific examples of structural proteins include keratin, collagen, elastin, arthropod elastin, silk and spider silk, and proteins derived from them.

[0085] Proteins derived from spider silk or silkworm silk that are filamentous proteins can be listed, for example, including Formula 1: [(A)] n Motif-REP m The represented domain sequence of the protein (here, in Equation 1, (A)). n A motif represents an amino acid sequence consisting of 4 to 20 amino acid residues, and (A) n The alanine residues in the motif constitute more than 80% of the total number of amino acid residues. REP represents an amino acid sequence consisting of 10–200 amino acid residues. m represents an integer from 8 to 300. Two or more (A) residues exist. n Motifs can have the same or different amino acid sequences. Two or more existing REPs can have the same or different amino acid sequences. Specifically, examples include proteins containing the amino acid sequences represented by sequence numbers 1 (PRT410), 2 (PRT853), 3 (PRT647), 4 (PRT699), and 5 (PRT698). These proteins have hydrophilic indices of -0.81, -0.68, 0.04, 0.17, and 0.43, respectively. The hydrophilic index values ​​are calculated according to the method described in International Publication No. 2014 / 103846.

[0086] Proteins that are sources of collagen can be listed, for example, those containing formula 2: [REP2] o The represented domain sequence is a protein (in Equation 2, o represents an integer from 5 to 300. REP2 represents an amino acid sequence consisting of Gly-XY, where X and Y represent any amino acid residue other than Gly. Two or more REP2 sequences can be the same or different amino acid sequences). Specifically, a protein containing the amino acid sequence represented by Serial Number 6 (Type IV collagen-Kai) can be listed. The amino acid sequence represented by Serial Number 6 is obtained by appending the amino acid sequence represented by Serial Number 10 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequence corresponding to the repeat portion and motif from residues 301 to 540 of the partial sequence of human Type IV collagen obtained from the NCBI database (NCBI GenBank accession number: CAA56335.1, GI: 3702452). The hydrophilicity index of Type IV collagen-Kai is -0.75.

[0087] Proteins that are sources of arthropod elastin can be listed, for example, those containing Formula 3: [REP3] p Proteins containing the domain sequence represented by the formula (here, in Formula 3, p represents an integer from 4 to 300. REP3 represents the amino acid sequence Ser-JJ-Tyr-Gly-U-Pro. J represents any amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Asp, Ser, and Thr. U represents any amino acid residue, particularly preferably an amino acid residue selected from the group consisting of Pro, Ala, Thr, and Ser. Two or more REP3s may have the same amino acid sequence or different amino acid sequences.). Specifically, proteins containing the amino acid sequence represented by sequence number 7 can be listed. The amino acid sequence represented by Serial No. 7 is obtained by replacing Thr at residue 87 with Ser and Asn at residue 95 with Asp in the amino acid sequence of arthropod elastin (NCBI GenBank accession number NP_611157.1, GL: 24654243). The amino acid sequence from residue 19 to residue 321 of the resulting sequence is then appended to the N-terminus with the amino acid sequence represented by Serial No. 10 (tag sequence and hinge sequence). The hydrophilicity index of arthropod elastin-Kai (Serial No. 7) is -1.22.

[0088] Proteins derived from elastin can be listed as having amino acid sequences such as those with NCBI GenBank accession numbers AAC98395 (human), I47076 (sheep), and NP786966 (bovine). Specifically, proteins containing the amino acid sequence represented by sequence number 8 can be listed. The amino acid sequence represented by sequence number 8 is obtained by appending the amino acid sequence represented by sequence number 10 (tag sequence and hinge sequence) to the N-terminus of the amino acid sequence from residue 121 to residue 390 of the amino acid sequence in NCBI GenBank accession number AAC98395. The hydrophilicity index of elastinshort (sequence number 8) is 0.42.

[0089] Proteins derived from keratin include, for example, type I keratin from goats (Caprahircus). Specifically, proteins containing the amino acid sequence represented by sequence number 9 (the amino acid sequence of NCBI GenBank accession number ACY30466) can be listed. Type I keratin 26 (sequence number 9) has a hydrophilicity index of -0.53.

[0090] (Recombinant cells)

[0091] The recombinant cells in this embodiment are recombinant cells that express recombinant proteins in the form of insoluble bodies, and can be obtained using general methods utilizing genetic engineering techniques.

[0092] Recombinant cells can be obtained, for example, by transforming a host (host cell) with an expression vector containing a nucleic acid sequence encoding a target protein and one or more regulatory sequences linked to that nucleic acid sequence in a functional manner.

[0093] Regulatory sequences are sequences that regulate the expression of recombinant proteins in the host (e.g., promoters, enhancers, ribosome-binding sequences, transcription termination sequences, etc.), and can be appropriately selected according to the type of host. Expression vectors can be appropriately selected according to the type of host, such as plasmid vectors, viral vectors, cosmid vectors, fosmid vectors, artificial chromosome vectors, etc.

[0094] As hosts, prokaryotes, as well as eukaryotes such as yeast, filamentous fungi, insect cells, animal cells, and plant cells, are all preferred. Bacteria, yeast, filamentous fungi, insect cells, plant cells, and animal cells are more preferred. For example, preferred examples of prokaryotes include Escherichia coli, Bacillus subtilis, Pseudomonas, Corynebacterium, and Lactococcus, and even more preferred examples include Escherichia coli cells.

[0095] As expression vectors, it is preferable to use expression vectors that can replicate autonomously in host cells or integrate into the host chromosome, and that contain a promoter at a location capable of transcribing the nucleic acid encoding the target protein. These vectors may contain ribosome-binding sequences, transcription termination sequences, or gene sequences that regulate the promoter.

[0096] As a promoter, any inducible promoter that functions in the host cell and can induce the expression of the target protein is acceptable. An inducible promoter is a promoter that can regulate transcription based on the presence of an inducing substance (expression inducer), the absence of a repressor molecule, or physical factors such as temperature, osmotic pressure, or pH increase or decrease.

[0097] As hosts for prokaryotes, microorganisms belonging to the genera Escherichia, Bacillus, Serratia, Bacillus, Microbacterium, Brachybacterium, Corynebacterium, and Pseudomonas can be listed.

[0098] As microorganisms belonging to the genus Escherichia, examples include Escherichia coli BL21 (Novagen), Escherichia coli BL21(DE3) (Life Technology), Escherichia coli BLR(DE3) (Merck Millipore), Escherichia coli DH1, Escherichia coli GI698, Escherichia coli HB101, Escherichia coli JM109, Escherichia coli K5 (ATCC23506), Escherichia coli KY3276, Escherichia coli MC1000, Escherichia coli MG1655 (ATCC47076), Escherichia coli No.49, Escherichia coli Rosetta (DE3) (Novagen), Escherichia coli TB1, Escherichia coli Turner (Novagen), Escherichia coli Turner (DE3) (Novagen), Escherichia coli W1485, Escherichia coli W3110 (ATCC27325), Escherichia coli (Escherichiacoli) XL1-Blue, and Escherichia coli XL2-Blue, etc.

[0099] Microorganisms belonging to the genus *Brachys* can be listed as follows: *Brachys stolonifer*, *Brachys potstanensis*, *Brachys mesospora*, *Brachys brevis*, *Brachys davidii*, *Brachys lateralis*, *Brachys swamp*, *Brachys parabrachys*, *Brachys reuteri*, *Brachys thermoplastia*, *Brachys brevis* 47 (FERMBP-1223), *Brachys brevis* 47K (FERMBP-2308), *Brachys brevis* 47-5 (FERMBP-1664), *Brachys brevis* 47-5Q (JCM8975), *Brachys davidii* HPD31 (FERMBP-1087), *Brachys davidii* HPD31-S (FERMBP-6623), *Brachys davidii* HPD31-OK (FERMBP-4573), and *Brachys davidii* SP3 strain (manufactured by Takara).

[0100] Microorganisms belonging to the genus Serratia include, for example, Serratia liquefacience ATCC14460, Serratia entomophila, Serratia aficaria, Serratia fonticola, Serratia grimesii, Serratia proteamaculans, Serratia odorifera, Serratia aplymuthica, and Serratia rubidaea.

[0101] Examples of microorganisms belonging to the genus Bacillus include Bacillus subtilis and Bacillus amyloliquefaciens.

[0102] Microorganisms belonging to the genus Microbe can be listed as, for example, Microbea ammoniac ATCC15354.

[0103] Examples of microorganisms belonging to the genus *Brevibacterium* include: *Brevibacterium formicatum* ATCC14020, *Brevibacterium glutamicum* ATCC13826, ATCC14067, *Brevibacterium immariophilum* ATCC14068, *Brevibacterium glutamicum* ATCC13869 ATCC13665, ATCC13869, *Brevibacterium roseum* ATCC13825, *Brevibacterium saccharolyticum* ATCC14066, *Brevibacterium sulfurogenum* ATCC19240, *Brevibacterium whiteum* ATCC15111, and *Brevibacterium cereus* ATCC15112.

[0104] Examples of microorganisms belonging to the genus Corynebacterium include: Corynebacterium ammoniagenes ATCC6871, ATCC6872; Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067; Corynebacterium acetoacidophilum ATCC13870; Corynebacterium acetoacidophilum ATCC15806; Corynebacterium alkylate ATCC21511; Corynebacterium argentiflorum ATCC15991; Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060; Corynebacterium lilyum ATCC15990; Corynebacterium molasses ATCC17965; Corynebacterium thermophilum ammoniagenes AJ12340 (FERMBP-1539); and Corynebacterium luxuriae ATCC13868.

[0105] Microorganisms belonging to the genus Pseudomonas include, for example, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas brassicacearum, Pseudomonas fulva, and Pseudomonas sp. D-0110.

[0106] As a method for introducing expression vectors into the host cells of the aforementioned prokaryotes, any method that introduces DNA into the host cells can be used. Examples include the method using calcium ions [Proc.Natl.Acad.Sci.USA,69,2110(1972)], the protoplast method (Japanese Patent Application Publication No. 63-248394), or the methods described in Gene,17,107(1982), Molecular & General Genetics,168,111(1979).

[0107] Transformation of microorganisms belonging to the genus *Brucea* can be carried out using methods such as those of Takahashi et al. (J. Bacteriol., 1983, 156: 1130-1134), Takagi et al. (Agric. Biol. Chem., 1989, 53: 3099-3100), or Okamoto et al. (Biosci. Biotechnol. Biochem., 1997, 61: 202-203).

[0108] Examples of vectors (hereinafter referred to as "vectors") used to introduce nucleic acids encoding target proteins include pBTrp2, pBTac1, pBTac2 (all sold by Boehringer Mannheim), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Patent Application Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], and pBluescriptIISK(-)(S (manufactured by Tratagene), pTrs30 [prepared from Escherichia coli JM109 / pTrS30 (FERMBP-5407)], pTrs32 [prepared from Escherichia coli JM109 / pTrS32 (FERMBP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERMB-400), Japanese Patent Application Publication No. 60-221091], pGKA2 [prepared from Escherichia coli IGKA2 (FERMBP-5408)], pTrs30 [prepared from Escherichia coli JM109 / pTrS30 (FERMBP-5407)], pGHA2 [prepared from Escherichia coli IGKA2 (FERMBP-5408)], pGKA ...]. Preparation of BP-6798, Japanese Patent Application Publication No. 60-221091, pTerm2 (US4686191, US4939094, US5160735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (Pharmacia), pET system (Novagen), etc.

[0109] When using Escherichia coli as a host, pUC18, pBluescriptII, pSupex, pET22b, and pCold are among the preferred vectors.

[0110] Specific examples of preferred vectors for microorganisms belonging to the genus *Brucea* include pUB110, pHY500 (Japanese Patent Application Publication No. 2-31682), pNY700 (Japanese Patent Application Publication No. 4-278091), pHY4831 (J. Bacteriol., 1987, 1239-1245), pNU200 (Shigezo Utaka, Journal of the Japanese Society for Agricultural Chemistry, 1987, 61: 669-676), pNU100 (Appl. Microbiol. Biotechnol., 1989, 30: 75-80), and pNU211 (J. Biochem., 1992, 112: 488-491). pNU211R2L5 (Japanese Patent Application Publication No. 7-170984), pNH301 (Appl. Environ. Microbiol., 1992, 58: 525-531), pNH326, pNH400 (J. Bacteriol., 1995, 177: 745-749), pHT210 (Japanese Patent Application Publication No. 6-133782), pHT110R2L5 (Appl. Microbiol. Biotechnol., 1994, 42: 358-363), or pNCO2, a shuttle vector of Escherichia coli and microorganisms belonging to the genus Bacillus (Japanese Patent Application Publication No. 2002-238569), etc.

[0111] As a promoter used in prokaryotes, there are no restrictions as long as it functions within the host cell. Examples of promoters derived from E. coli or bacteriophages include the trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, and T7 promoter. Additionally, promoters that have been artificially designed and modified, such as promoters consisting of two Ptrp molecules tandemly (Ptrp×2), tac promoters, lacT7 promoters, and letI promoters, can also be used. Plasmids with the Shine-Dalgarno sequence (which serves as the ribosome binding sequence) positioned at an appropriate distance (e.g., 6–18 bases) from the start codon are preferred. In the expression vectors described above, the expression of the nucleic acid does not necessarily require a transcription termination sequence, but it is preferable to place the transcription termination sequence immediately downstream of the nucleic acid encoding the target protein.

[0112] Examples of hosts for eukaryotes include yeast, filamentous fungi (molds, etc.), and insect cells.

[0113] Examples of yeasts include those belonging to the genera *Saccharomyces*, *Schizosaccharomyces*, *Kluyveromyces*, *Trichosporon*, *Schwanniomyces*, *Pichia*, *Candida*, *Yersinia*, and *Hansenula*. More specifically, examples include *Saccharomyces cerevisiae*, *Schizosaccharomyces pombe*, *Kluyveromyces lactis*, *Kluyveromyces marxianus*, *Trichosporon pullullans*, *Schwanniomyces alluvius*, *Schwanniomyces occidentalis*, *Candida utilis*, *Pichiapastoris*, *Pichia angusta*, *Pichiamethanolica*, *Pichia polymorpha*, *Pichiastipitis*, *Yarrowialipolytica*, and *Hansenula polymorpha*.

[0114] Expression vectors used when yeast is the host cell typically preferably contain an origin of replication (in cases where amplification in the host is required) and selection markers for vector proliferation in E. coli, promoters and terminators for recombinant protein expression in yeast, and selection markers for yeast.

[0115] In the case of a non-integrating expression vector, it is preferable to further include an autonomously replicating sequence (ARS). This can improve the stability of the expression vector in cells (Myers, AM, et al. (1986) Gene45:299-310).

[0116] Examples of vectors that use yeast as a host include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), YIp, pHS19, pHS15, pA0804, pHIL3Ol, pHIL-S1, pPIC9K, pPICZα, pGAPZα, and pPICZB.

[0117] As a specific example of a promoter in the case of yeast as the host, there are no restrictions as long as it can be expressed in yeast. Examples include promoters of genes related to glycolysis systems such as hexokinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock peptide promoter, MFα1 promoter, CUP1 promoter, pGAP promoter, pGCW14 promoter, AOX1 promoter, MOX promoter, etc.

[0118] As a method for introducing expression vectors into yeast, any method that introduces DNA into yeast can be used, such as electroporation (MethodsEnzymol., 194, 182 (1990)), protoplast method (Proc.Natl.Acad.Sci., USA, 81, 4889 (1984)), lithium acetate method (J.Bacteriol., 153, 163 (1983)), and the method described in Proc.Natl.Acad.Sci.USA, 75, 1929 (1978).

[0119] Examples of filamentous fungi include those belonging to the genera *Acremonium*, *Aspergillus*, *Ustilago*, *Trichoderma*, *Neurospora*, *Fusarium*, *Humicola*, *Penicillium*, *Myceliophtora*, *Botryts*, *Magnaporthe*, *Mucor*, *Metarhizium*, *Monascus*, *Rhizopus*, and *Rhizopus*.

[0120] Specific examples of filamentous fungi include *Acremonium alabamaense*, *Acremonium cellulolyticus*, *Aspergillus aculeatus*, *Aspergillus awamori*, *Aspergillus oryzae*, *Aspergillus sake*, *Aspergillus sojae*, *Aspergillus tubigensis*, and *Aspergillus niger*. Aspergillus nidulans, Aspergillus parasiticus, Aspergillus ficuum, Aspergillus sphoeicus, Aspergillus foetidus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus japonicus, Trichoderma maviride, Trichoderma harzianum Trichoderma mareseei, Chrysosporium lucknowense, Thermoascus, Sporotrichum, Sporotrichum cellulophilum, Talaromyces, Thievaria terrestris, Thievaria, Neurosporacrassa, Fusarium oxysporus Fusarium graminearum, Fusarium venenatum, Humicolainsolens, Penicillium chrysogenum, Penicillium camemberti, Penicillium canescens, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseumThe fungi include *Penicillium purpurogenum*, *Penicillium roqueforti*, *Myceliophta orathermophilum*, *Mucorambiguus*, *Mucorcircinelloides*, *Mucorfragilis*, *Mucorhiemalis*, *Mucorina equisporus*, *Mucoroblongi ellipticus*, *Mucorracemosus*, *Mucorrecurvus*, *Mocorsaturninus*, *Mocorsubtilissmus*, *Ogataea polymorpha*, *Phanerochaetechrysosporium*, *Rhizomucormiehei*, *Rhizomucorpusillus*, and *Rhizopusarrhizus*.

[0121] Specific examples of promoters in the case of filamentous fungi as hosts can be any of the following: genes involved in the glycolysis system, genes involved in constitutive expression, enzyme genes involved in hydrolysis, etc. Specifically, amyB, glaA, agdA, glaB, TEF1, xynF1tannasegene, No.8AN, gpdA, pgkA, enoA, melO, sodM, catA, catB, etc.

[0122] The introduction of expression vectors into filamentous fungi can be performed using existing, well-known methods. Examples include, for instance, the method used by Cohen et al. (calcium chloride method) [Proc. Natl. Acad. Sci. USA, 69:2110 (1972)], the protoplast method [Mol. Gen. Genet., 168:111 (1979)], the competent cell method [J. Mol. Biol., 56:209 (1971)], and electroporation.

[0123] Examples of insect cells include, for instance, cells from lepidopteran insects. More specifically, examples include insect cells from Spodopterafrugiperda such as Sf9 and Sf21, and insect cells from Trichoplusiani such as High5.

[0124] Examples of vectors used when insect cells are used as hosts include baculoviruses such as the alfalfa silver-striped noctuid moth nucleopolyhedrosis virus (Autographacalifornicanuclearpolyhedrosisvirus, A Laboratory Manual, WH Freeman and Company, New York (1992)).

[0125] When using insect cells as a host, peptide expression can be performed using methods described in, for example, Current Protocols in Molecular Biology; Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992); Bio / Technology, 6, 47 (1988). Specifically, a recombinant gene vector and a baculovirus can be co-introduced into insect cells. After obtaining the recombinant virus (expression vector) in the insect cell culture supernatant, the recombinant virus is further used to infect the insect cells, thereby expressing the peptide. Examples of gene vectors used in this method include pVL1392, pVL1393, and pBlueBacIII (all manufactured by Invitorogen).

[0126] Examples of methods for preparing recombinant viruses, recombinant gene delivery vectors, and co-introduction of baculoviruses into insect cells include the calcium phosphate method (Japanese Patent Application Laid-Open No. 2-227075) and the liposome transfection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)).

[0127] The recombinant vectors described above preferably further contain selection marker genes for transformant selection. For example, in *E. coli*, resistance genes against various agents such as tetracycline, ampicillin, and kanamycin can be used as selection marker genes. Alternatively, selection markers that complement gene variations related to nutritional requirements can be used. In yeast, resistance genes against hexamethylenetetramine can be used as selection marker genes, as well as selection markers such as genes that complement gene variations related to nutritional requirements, LEU2, URA3, TRP1, and HIS3. In filamentous fungi, genes selected as selection markers include niaD (Biosci. Biotechnol. Biochem., 59, 1795-1797 (1995)), argB (Enzyme Microbiol Technol, 6, 386-389, (1984)), sC (Gene, 84, 329-334, (1989)), ptrA (Biosci. Biotechnol Biochem, 64, 1416-1421, (2000)), and pyrG (Bioch. Biotechnol Biochem., 64, 1416-1421, (2000)). The gene sequence consists of marker genes from the following groups: emBiophysResCommun, 112, 284-289 (1983), amdS (Gene, 26, 205-221 (1983)), azadirachtin resistance gene (MolGenGenet, 261, 290-296 (1999)), benomyl resistance gene (ProcNatlAcadSciUSA, 83, 4869-4873 (1986)), and hygromycin resistance gene (Gene, 57, 21-26 (1987)), as well as leucine requirement complement genes. Additionally, when the host is a nutrient requirement variant, wild-type genes complementary to that nutrient requirement can also be used as selection marker genes.

[0128] The selection of hosts transformed using the above expression vector can be performed through plaque hybridization and colony hybridization using probes that selectively bind to the above nucleic acids. As such probes, probes obtained by modifying a portion of the DNA fragment amplified by PCR based on the sequence information of the above nucleic acids with a radioactive isotope or digoxigenin can be used.

[0129] (Expression of recombinant proteins)

[0130] In recombinant cells transformed with the aforementioned expression vector for expressing the target protein, the recombinant protein is expressed intracellularly in an insoluble form. The recombinant protein can be expressed by culturing the recombinant cells in a culture medium. The method for culturing the recombinant cells in the culture medium can follow the methods commonly used in host cell culture.

[0131] When the host is a prokaryote such as Escherichia coli or a eukaryote such as yeast, any culture medium, whether natural or synthetic, can be used as long as it contains carbon sources, nitrogen sources, and inorganic salts that the host can assimilate and can efficiently culture the host.

[0132] As a carbon source, any carbon source that the host can assimilate can be used, such as carbohydrates such as glucose, fructose, sucrose and molasses containing these sugars, starch and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

[0133] As nitrogen sources, ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermented microorganisms and their digestion products can be used.

[0134] As inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate can be used.

[0135] The culture of prokaryotes such as Escherichia coli or eukaryotes such as yeast can be carried out under aerobic conditions, such as shaking culture or deep aeration and stirring culture. The culture temperature is, for example, 15–40°C. The culture time is usually 16 hours to 7 days. The pH of the culture medium is preferably maintained between 3.0 and 9.0. The pH of the culture medium can be adjusted using inorganic acids, organic acids, alkaline solutions, urea, calcium carbonate, and ammonia.

[0136] In addition, antibiotics such as ampicillin and tetracycline can be added to the culture medium as needed during cultivation. When culturing microorganisms transformed using expression vectors with inducible promoters, inducers can be added to the culture medium as needed. For example, when culturing microorganisms transformed using expression vectors with lac promoters, isopropyl-β-D-thiogalactopyranoside can be added to the culture medium; when culturing microorganisms transformed using expression vectors with trp promoters, indoleacrylic acid can be added to the culture medium.

[0137] As a culture medium for insect cells, commonly used TNM-FH medium (manufactured by Pharmingen), Sf-900 II FM medium (manufactured by Life Technologies), ExCell400, ExCell405 (both manufactured by JRH Biosciences), and Grace insect medium (Nature, 195, 788 (1962)) can be used.

[0138] Insect cells can be cultured for 1 to 5 days under conditions such as a culture medium pH of 6–7 and a culture temperature of 25–30°C. Additionally, antibiotics such as gentamicin can be added to the culture medium as needed during the culture process.

[0139] When the host is a plant cell, the transformed plant cell can be cultured directly, or it can differentiate into a plant organ for culture. Commonly used culture media for these plant cells include Murashige & Skoog (MS) medium, White medium, or media prepared by adding plant hormones such as auxin and cytokinin to these media.

[0140] Animal cell culture can be carried out for 3 to 60 days under conditions such as a culture medium pH of 5–9 and a culture temperature of 20–40°C. Additionally, antibiotics such as kanamycin and hygromycin can be added to the culture medium as needed during the culture process.

[0141] Using the above method, the target protein can be expressed in recombinant cells in an insoluble form.

[0142] (A) The disruption steps of recombinant cells

[0143] Step (A) is to lyse recombinant cells that express the target recombinant protein in the form of insoluble bodies to obtain a lysed suspension containing the insoluble bodies of the recombinant protein.

[0144] The disruption of recombinant cells can be carried out using known methods. These include cell disruption by enzymatic treatment with lysozyme, mutalysin, lysozyme, yeast lysin, etc.; cell disruption by contact with organic solvents, etc.; cell disruption by osmotic pressure; physical / mechanical cell disruption such as ball milling, Freund's crusher, high-pressure homogenizer, and ultrasonic treatment; and combinations thereof.

[0145] The culture medium obtained through the above culture can be used directly in the disruption of recombinant cells, but in order to improve the purity of the recombinant protein obtained later, it is preferable to use a suspension of washed recombinant cells.

[0146] The suspension of washed recombinant cells can be prepared using the following method: Recombinant cells are separated from the culture medium by centrifugation, filtration, etc. Considering subsequent steps, washing with water is preferred, and washing with a buffered aqueous solution followed by further washing with water is also preferred. Alternatively, the recombinant cells can be prepared by suspending them at an appropriate concentration in a solution suitable for the above-described disruption method.

[0147] Alternatively, recombinant cells obtained from the culture medium can be treated with organic solvents or the like, and the insoluble fractions, after removing the soluble fractions such as host-derived proteins from the recombinant cells, can be suspended at an appropriate concentration in a solution suitable for the above-described disruption method to obtain a suspension. In this case, when the bacterial cells are disrupted by treatment with organic solvents or the like, this treatment with organic solvents or the like (contact with organic solvents or the like) can be considered as the above-described disruption treatment. That is, the following (B) step of agglutination of recombinant protein insolubles can be performed after treatment with organic solvents or the like.

[0148] Suitable solutions include industrial water, deionized water, RO (reverse osmosis) water, and buffer solutions. Examples of buffer solutions include, for instance, Tris / HCl buffer solution.

[0149] The resulting fragmented suspension contains insoluble forms of the recombinant protein. In this specification, "insoluble form" refers to a protein that is insoluble in solution (suspension), and may sometimes form insoluble particles.

[0150] In addition, the ruptured suspension contains cell debris that can be easily separated by centrifugation, and the following coagulation step can be performed using the suspension after removing these cell debris.

[0151] In addition, sometimes insoluble particles in the broken suspension are mixed with impurities, which can be separated by centrifugation. In this case, centrifugation can be performed, and the resulting precipitate fraction containing insoluble particles can be resuspended in the above-mentioned buffer aqueous solution for further coagulation.

[0152] (B) Agglutination steps of recombinant protein insolubles

[0153] Step (B) involves adding one or more ingredients selected from the group consisting of metal salts, acids, and anionic coagulants to the broken suspension obtained in step (A) above, causing the insoluble parts of the recombinant protein to agglomerate, thereby obtaining a recombinant protein agglomerate. In step (B), heating and / or stirring may be performed as needed.

[0154] Examples of metal salts include alkaline earth metal salts and earth metal salts (earth metal salts). Specifically, examples include alkaline earth metal halides, alkaline earth metal nitrates, alkaline earth metal sulfates, earth metal halides, earth metal nitrates, and earth metal sulfates. Among metal salts, polyvalent metal salts with a valence of two or more are preferred.

[0155] Examples of alkaline earth metal halides include calcium chloride, magnesium chloride, magnesium bromide, calcium bromide, magnesium iodide, and calcium iodide.

[0156] Examples of alkaline earth metal nitrates include calcium nitrate, magnesium nitrate, strontium nitrate, and barium nitrate.

[0157] Examples of alkaline earth metal sulfates include calcium sulfate, magnesium sulfate, strontium sulfate, and barium sulfate.

[0158] Examples of earth metal halides include aluminum trichloride and gallium trichloride.

[0159] Examples of earth metal nitrates include aluminum nitrate and gallium nitrate.

[0160] Examples of earth metal sulfates include aluminum sulfate and gallium sulfate.

[0161] These metal salts can be used individually or in combination of two or more.

[0162] Preferred metal salts include alkali metal halides and alkaline earth metal halides. Specific preferred examples include lithium chloride and calcium chloride.

[0163] Regarding the amount of metal salt added, if the recombinant protein forms dense, insoluble particles in the lysed suspension, even a small amount can be effective, and the metal salt can be added at a level of, for example, 0.01–20 mM, preferably 1–10 mM. If the insoluble body does not form insoluble particles, or if the insoluble particles require time to settle through centrifugation, the metal salt can be added at a level of 2–50 mM, preferably 5–10 mM.

[0164] As the acid, either inorganic or organic acids can be used. Preferred acids include oxyacids, etc.

[0165] Examples of oxyacids that are inorganic acids include sulfuric acid, nitric acid, and phosphoric acid. Examples of oxyacids that are organic acids include formic acid, acetic acid, citric acid, and tartaric acid. Acetic acid, sulfuric acid, and citric acid are preferred as oxyacids, with citric acid being more preferred.

[0166] Regarding the amount of acid added, if the recombinant protein forms dense, insoluble particles in the lysed suspension, even a small amount can be effective, and the acid can be added at a level of, for example, 0.01–20 mM, preferably 1–20 mM, and more preferably 5–20 mM. If the protein is an insoluble mass that has not formed insoluble particles, or if the insoluble particles require time to settle through centrifugation, the acid can be added at a level of 2–50 mM, preferably 10–30 mM.

[0167] These acids can be used individually or in combination of two or more.

[0168] In this specification, "anionic flocculant" refers to a polymeric flocculant (polymer) containing organic anionic groups. Examples of anionic flocculants include polyacrylates, anionic polyacrylamides, and acrylamide-acrylate copolymers. Specifically, examples include Kurita Kogyo Co., Ltd.'s Clift PA series (PA-923, PA-896, PA-895, PA-893, PA-865, PA-823, PA-813, PA-804, PA-465, PA-404, PA-402, PA-265, etc.), Mitsui Chemicals Acrylica Polymer Co., Ltd.'s Akoflok (A-95~A-100, A-110~A-150, A-190, A-235H~A-250, etc.), and Smiflok (…). FA-40~FA-70), Mitsubishi Rayon Co., Ltd.'s ダイヤフロック AP series (AP335B, AP741B, AP825C, etc.), Taiki Chemical Co., Ltd.'s タキフロックA series (A-102~A-106, A-108, A-142, A-162), Togami Electric Manufacturing Co., Ltd.'s Togami Electric Co., Ltd. (TA-089, TA-104, TA-109, TA-124, TA-144, TAE-2325, TAE-2335, TAE-2644, etc.), etc.

[0169] These coagulants also have the effect of causing host cells to aggregate. Therefore, when using coagulants, it is preferable to use a fragmented suspension from which cell debris has been removed beforehand. The amount of anionic coagulant added is such that the recombinant protein in the fragmented suspension is 0.001–0.1%, preferably 0.01–0.05%.

[0170] Regarding the addition of metal salts, acids, or anionic coagulants, the combined addition at low concentrations is more effective than adding them individually.

[0171] In the agglomeration step (B), after adding one or more selected from the group consisting of metal salts, acids, and anionic coagulants, heating may be performed to promote agglomeration and make the aggregates larger. The heating method is not particularly limited. The heating temperature (peak temperature) is not particularly limited; from the viewpoint of efficiently obtaining insoluble bodies or insoluble particles and from the viewpoint of cell inactivation, depending on the type of the target recombinant protein, the heating temperature is, for example, 60°C or higher, preferably 70°C or higher, and more preferably 80°C or higher. Furthermore, from the viewpoint of inhibiting the decomposition of the target protein and improving its purity, depending on the type of target protein, the temperature is, for example, 130°C or lower, preferably 110°C or lower, and more preferably 90°C or lower.

[0172] The heating time (the time to maintain the heating temperature) is not particularly limited. From the viewpoints of efficiently obtaining insoluble bodies or insoluble particles and from the viewpoints of bacterial cell inactivation, depending on the type of recombinant protein, it is, for example, 0.5 hours or more, preferably 1 hour or more, and more preferably 2 hours or more. Furthermore, from the viewpoint of inhibiting the decomposition of the target protein and improving work efficiency, depending on the type of target protein, it is, for example, 15 hours or less, preferably 10 hours or less, and more preferably 5 hours or less.

[0173] The heating time for obtaining insoluble bodies or insoluble particles can be significantly shortened by continuously heating the broken suspension. The temperature for continuous heating of the broken suspension is not particularly limited; however, from the viewpoints of efficiently obtaining insoluble bodies or insoluble particles and cell inactivation, the heating temperature is, for example, 70°C or higher, preferably 80°C or higher, and more preferably 90°C or higher, depending on the type of recombinant protein. Furthermore, from the viewpoint of inhibiting the decomposition of the target protein and improving its purity, the temperature is, for example, 140°C or lower, preferably 120°C or lower, and more preferably 100°C or lower, depending on the type of target protein.

[0174] When continuously heating the broken suspension, the heating time for obtaining insoluble bodies or insoluble particles is not particularly limited. From the viewpoints of efficiently obtaining insoluble bodies or insoluble particles and from the viewpoints of cell inactivation, the heating time is, for example, 1 second or more, preferably 10 seconds or more, and more preferably 30 seconds or more, depending on the type of the target recombinant protein. Furthermore, from the viewpoint of inhibiting the decomposition of the target protein and improving work efficiency, the heating time is, for example, 120 seconds or less, preferably 90 seconds or less, and more preferably 60 seconds or less, depending on the type of the target protein.

[0175] There are no particular limitations on the method of continuously heating the broken suspension, as long as the insoluble body or insoluble particles can be heated to above 70°C and below 140°C and the heated temperature can be maintained for less than 120 seconds. Methods such as using a liquid continuous sterilization device can be cited, and in particular, the liquid continuous sterilization device MINIUHTT-20 (manufactured by Powerpoint International) can be cited.

[0176] In the agglomeration step (B), to make the agglomerates larger, further stirring can be performed on the basis of heating. The means of stirring is not particularly limited. The stirring speed is not particularly limited, but from the viewpoint of efficiently obtaining insoluble particles, a speed at which the agglomerates of insoluble matter in the solution do not precipitate is preferred, for example, 70 rpm or more, preferably 150 rpm or more, and more preferably 300 rpm or more. Furthermore, from the viewpoint of suppressing the breakage of the formed insoluble matter, a speed of 1500 rpm or less, preferably 1000 rpm or less, and more preferably 500 rpm or less is preferred. Regarding the stirring time, it can be performed at any time between the agglomeration steps in (B), but if heating is performed, it is preferable to perform the stirring simultaneously with heating.

[0177] (C) Separation steps of recombinant protein condensates

[0178] Step (C) is the step of separating the aggregates obtained in step (B) from the suspension. Aggregation of the recombinant protein insolubles is initiated simultaneously with the addition of one or more selected from the group consisting of metal salts, acids, and anionic flocculants to the broken suspension. The aggregates can then be separated using appropriate separation methods, such as natural sedimentation, centrifugation, or filtration. After adding one or more selected from the group consisting of metal salts, acids, and anionic flocculants, heating and further stirring as needed promote aggregation, resulting in larger aggregates that are easier to separate.

[0179] In one example, the aggregates could be recovered by centrifugation at 2500×g for 5–30 minutes. When recombinant proteins form insoluble particles in the broken suspension that are already centrifugally separable, one or more of a group consisting of metal salts, acids, and anionic flocculants can be added, followed by heating and stirring as needed, thereby further enlarging the insoluble particles and causing them to settle naturally. For recombinant protein insolubles that are difficult to separate by centrifugation, one or more of a group consisting of metal salts, acids, and anionic flocculants can be added, followed by heating and stirring as needed, further enlarging the insoluble particles, and then separating them by centrifugation, filtration, etc.

[0180] To date, there have been no reports of centrifugation forces as low as 2500 × g capable of settling insoluble particles; typically, cylindrical centrifuges with centrifugal forces exceeding 12000 × g are used. However, according to the present invention, insoluble recombinant proteins can be settled in aggregate form with even lower centrifugal forces. This means that plate (disc) centrifuges, decanter centrifuges, and other centrifuges such as the Westfalia centrifuge, clarifier, and Alfa Laval centrifuge, which have been used only for bacterial cell separation until now, as well as basket centrifuges, can be used for the separation of insoluble recombinant proteins. These plate and decanter centrifuges have centrifugal forces below 10000 × g and can continuously separate large volumes of suspensions, thus becoming extremely useful tools in industrial production.

[0181] Furthermore, even large, insoluble particles that can easily settle by conventional centrifugation are highly likely to cause clogging in membrane filtration without the aforementioned coagulation step. However, by performing this coagulation step, membrane separation can be easily achieved. In particular, by adding one or more of a group consisting of metal salts, acids, and anionic coagulants, followed by heating and stirring as needed, membrane separation can be performed even more easily.

[0182] Furthermore, host cell-derived impurities that can be separated from the recombinant protein insolubles can be removed simply through the separation process, thereby improving the purity of the recombinant protein. Additionally, if the separated recombinant protein insolubles are resuspended and subjected to further aggregation and separation steps, the purity can be further improved.

[0183] The particle size of the recombinant protein chondrite obtained by the separation step (C) can be determined using, for example, the inductively coupled plasma method. From the viewpoint of improving filterability, the particle size of the recombinant protein chondrite is, for example, 4 μm or more, preferably 5 μm or more, more preferably 10 μm or more, and even more preferably 15 μm or more. Furthermore, there is no particular upper limit to the particle size of the chondrite; it can be 50 μm or less, 40 μm or less, 30 μm or less, or 20 μm or less.

[0184] As an inductive zone method, the particle size distribution determination method based on JIS Z8832 can be cited, and in particular, the determination method using the particle size number analyzer CDA-1000 (Sysmex Corporation) can be cited.

[0185] The recombinant protein condensate obtained by separation can be further purified to improve its purity, for example, by using the method described in Japanese Patent Application Publication No. 2013-523665.

[0186] Example

[0187] The present invention will now be described in more detail with reference to embodiments. However, the present invention is not limited to the embodiments described below.

[0188] (1) Preparation of target protein expression line (recombinant cell)

[0189] Nucleic acids, GEN495, GEN971, GEN740, GEN797, and GEN796, encoding filamentin proteins derived from spider silk and containing amino acid sequences represented by sequence numbers 1 (PRT410), 2 (PRT853), 3 (PRT647), 4 (PRT699), and 5 (PRT698), were synthesized. These nucleic acids had an NdeI site appended to the 5' end and an EcoRI site appended downstream of the stop codon. The hydrophilicity index (HI) and molecular weight of each protein are shown in Table 1.

[0190] [Table 1]

[0191] These five nucleic acids were cloned into the cloning vector (pUC118). Then, the nucleic acids were treated with restriction enzymes using NdeI and EcoRI, excised, and recombined into the protein expression vector pET-22b(+), yielding the expression vectors. These five expression vectors were then used to transform *E. coli* BLR(DE3) to obtain transformed *E. coli* (recombinant cells) expressing the target protein.

[0192] (2) Expression of the target protein

[0193] The transformed *E. coli* were cultured in 2 mL of LB medium containing ampicillin for 15 hours. This culture was then added to 100 mL of seed culture medium (Table 2) containing ampicillin to adjust the OD... 600 The OD value reached 0.005. The culture medium temperature was maintained at 30°C, and flask incubation was continued until the OD value reached 0.005. 600 Seed culture medium is obtained when the concentration reaches 5 (approximately 15 hours).

[0194] [Table 2]

[0195] The seed culture solution was added to a fermenter containing 500 mL of production medium (Table 3) to allow OD to... 600 The concentration was reduced to 0.05. The culture medium temperature was maintained at 37°C, and the pH was kept constant at 6.9. Additionally, the dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation point.

[0196] [Table 3]

[0197] After the glucose in the production medium was completely consumed, a feed solution (455 g / L glucose, 120 g / L yeast extract) was immediately added at a rate of 1 ml / min. The culture medium temperature was maintained at 37°C, and the pH was kept constant at 6.9. The dissolved oxygen concentration in the culture medium was maintained at 20% of the dissolved oxygen saturation point for 20 hours. Then, 1 M isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture medium to reach a final concentration of 1 mM, inducing the expression of the target protein. Twenty hours after the addition of IPTG, the culture medium was centrifuged, and the bacterial cells were recovered. SDS-PAGE was performed using bacterial cells prepared from the cultures before and after IPTG addition. The appearance of bands indicating the size of the target protein, dependent on IPTG addition, confirmed that the target protein was expressed in an insoluble form.

[0198] [One of the effects of adding metal salts in Example 1]

[0199] In an RO water suspension of *E. coli* BLR(DE3) expressing PRT853 (HI: -0.68) in insoluble form, 1.8 μg / g wet cell deoxyribonuclease (SIGMA-ALDRICH) and 164 μg / g wet cell lysozyme (Thermo Fisher Scientific) were added. The cells were homogenized four times at 600 bar at room temperature using a high-pressure homogenizer (GEA, Pandaplus) to disrupt the cells. After disruption, the cells were centrifuged (TOMYMX-305) at 11000 × g for 5 minutes to obtain the insoluble particles. These insoluble particles were relatively small particles obtained through centrifugation, which would otherwise require a considerable amount of time. After suspending these insoluble particles in water, the metal salts shown in Table 4 were added at a concentration of 0.5 M. Figure 1 These are photographs of the samples after adding metal salts and centrifuging at 2680×g for 10 seconds.

[0200] It was confirmed that by adding a polyvalent metal salt, the insoluble matter could be precipitated by centrifugation at 2680×g for 10 seconds.

[0201] [Table 4]

[0202] [Example 2: The Second Effect of Adding Metal Salts]

[0203] For the metal salts (magnesium chloride, calcium chloride, magnesium sulfate, and magnesium nitrate) that exhibited excellent agglomeration effects in Example 1, the agglomeration effect at low concentrations was confirmed using the insoluble form of PRT853 (see Table 5). Figure 2 These are photographs of the samples after adding metal salts and centrifuging at 2680×g for 10 seconds.

[0204] Aggregation was observed in all metal salts at 1 mM, but significant aggregation was observed at concentrations above 5 mM.

[0205] [Table 5]

[0206] [Example 3: Effects of HI on different proteins]

[0207] The effects of adding metal salts to proteins with different hydrophobicities were confirmed. Using the same method as in Example 1, the aggregation effects of adding metal salts (calcium chloride and magnesium chloride) to four insoluble proteins—PRT410 (HI: -0.81), PRT647 (HI: 0.04), PRT699 (HI: 0.17), and PRT698 (HI: 0.43)—were confirmed.

[0208] Deoxyribonuclease (1.8 μg / g wet cells) and lysozyme (164 μg / g wet cells) were added to an RO-water suspension of *E. coli* BLR(DE3) expressing various insoluble forms. The cells were homogenized four times at room temperature using an autoclave at 600 bar to disrupt the cell structure. After disruption, calcium chloride or magnesium chloride was added to the disrupted suspension at a concentration of 10–150 mM, and the suspension was centrifuged at 2680 × g for 10 seconds to confirm the aggregation status. Figure 3 These are photographs of the samples after adding metal salts and centrifuging at 2680×g for 10 seconds.

[0209] It was confirmed that, through this centrifugation process, the insoluble forms of PRT410 and PRT699 can precipitate without the addition of metal salts (calcium chloride and magnesium chloride), and the addition of even small amounts of metal salts can induce more concentrated precipitation. On the other hand, PRT647 and PRT698 cannot precipitate under these centrifugation conditions without the addition of metal salts, but the addition of metal salts can cause them to agglomerate and precipitate. In particular, higher concentrations result in more concentrated agglomeration and precipitation (see reference). Figure 3 ).

[0210] The effect of metal salt addition was confirmed in proteins with different hydrophobicities, therefore it is believed that this metal salt addition method can be applied to the aggregation of insoluble bodies of various proteins.

[0211] [Example 4: Improvement of Purification Quality]

[0212] The four insoluble compounds, PRT410, PRT647, PRT699, and PRT698, can be precipitated in 5 minutes by centrifugation at 11000×g and 20℃. The precipitate obtained by centrifugation is then resuspended in RO water to confirm the effect of adding metal salts to this suspension (centrifuged precipitate resuspension).

[0213] For insolubles resuspended in RO water, the effect of adding metal salts was confirmed in the same manner as in Example 3. Figure 4 These are photographs of the samples after centrifugation at 2680×g for 10 seconds following the addition of metal salts. Figure 4 As shown, the effect of adding metal salts was confirmed in the same way as in Example 3. Furthermore, as shown below, this resuspension process can improve the purity of the insoluble particles.

[0214] Figure 5 Table 6 shows the results of the addition of metal salts and the centrifugation precipitation and resuspension operation that resulted in improved purity of the insoluble material. Figure 5 These are photographs showing the results (electrophoresis results) of analyzing the various treatment solutions of PRT410 obtained in Examples 3 and 4 using SDS-PAGE. Figure 5 In lanes A and B, a suspension of PRT410 cells freshly broken using a high-pressure homogenizer (broken suspension) was applied to lane 1; in lane 2, a precipitate fraction obtained by adding calcium chloride to the broken suspension at a concentration of 10 mM and centrifuging at 2500 × g for 5 minutes was applied; in lane 3, a precipitate fraction obtained by adding magnesium chloride to the broken suspension at a concentration of 10 mM and centrifuging at 2500 × g for 5 minutes was applied; and a molecular weight marker protein was applied to lane M.

[0215] Figure 5 Photograph A was obtained after electrophoresis by staining with Oriole (trademark) fluorescent gel staining agent (manufactured by Bio-Rad) which can stain all proteins. Figure 5 Image B is obtained after electrophoresis using InVision (trademark) His-tagged intragel staining reagent (Thermo Fisher Scientific) that reacts with the His-tagged region of PRT410. PRT410, with a theoretical molecular weight of 53.6 kDa, was detected as a band close to the molecular weight marker of 60 kDa.

[0216] The electrophoretic bands of Oriole-stained gels were analyzed using a GelDoc EZ gel imaging system (manufactured by BIORAD) to calculate the purification purity of PRT410 in each treatment solution. The results are shown in Table 6 (freshly disrupted).

[0217] The purity of the precipitate fraction obtained without the addition of metal salts is 10.5%. However, by adding metal salts, it can be made to aggregate and precipitate densely. Therefore, adding calcium chloride can increase the purity to 30.7%, and adding magnesium chloride can increase the purity to 34.1% (reference). Figure 5 Lanes 1-3, Table 6 (just after breaking)).

[0218] in addition, Figure 5 In lanes A and B, the broken suspension (lane 1) was centrifuged at 11000×g, 20℃, for 5 minutes in lane 4 to settle, and the resulting precipitate fraction was resuspended in RO water to obtain a suspension (centrifuged precipitate resuspension). In lane 5, calcium chloride was added to this centrifuged precipitate resuspension at a concentration of 10 mM, and centrifuged at 2500×g for 5 minutes to obtain a precipitate fraction. In lane 6, magnesium chloride was added to this centrifuged precipitate resuspension at a concentration of 10 mM, and centrifuged at 2500×g for 5 minutes to obtain a precipitate fraction. The suspension, freshly broken up by the bacteria, was then centrifuged at 11000×g, 20℃, for 5 minutes using a high-pressure homogenizer to settle, and the resulting precipitate fraction was resuspended in RO water (centrifuged precipitate resuspension). This process increased the purity from 10.5% to 34.8% (reference). Figure 5 Lane 4 and Table 6 (centrifugal precipitate resuspension) show that by adding calcium chloride or magnesium chloride to the centrifuged precipitate resuspension at a concentration of 10 mM to cause the insoluble particles to agglomerate, separation can be achieved by low-speed centrifugation at 2500 × g for 5 minutes, and the purity of the obtained precipitate fractions is increased to 48.6% and 50.1%, respectively (reference). Figure 5 Lanes 5 and 6, Table 6 (centrifugation precipitation and resuspension) show that adding metal salts can significantly improve purity.

[0219] It can be confirmed that this method of adding metal salts is not only highly effective in the aggregation of insoluble bodies, but also an effective means of removing impurities from host cells.

[0220] [Table 6]

[0221] In addition, for the precipitates of four insoluble substances (PRT410, PRT647, PRT699, and PRT698) obtained by adding 50 mM metal salt to the centrifuged precipitates and resuspended and broken suspensions, followed by agglutination and centrifugation at 2680 × g for 10 seconds, the protein recovery rates were calculated. The results are shown in Table 7.

[0222] The recovery rate was calculated as follows: the absorbance at 595 nm was measured using an ELISA reader (TECAN, InfiniteF200). The absorbance value before centrifugation was set to 0%, and the absorbance value of the supernatant obtained after treatment with 11000×g for 10 minutes was set to 100%.

[0223] [Table 7]

[0224] Table 7 shows that by adding metal salts, insoluble substances can be recovered in extremely high yields.

[0225] It can be confirmed that this method of adding metal salts is an excellent method as follows: there is no limitation on the type of insoluble protein; in addition, regardless of whether the insoluble body exists in the form of dense insoluble particles, it is an extremely effective means of agglomerating insoluble bodies and removing impurities from host cells, and can recover insoluble bodies with a very high yield.

[0226] [One of the effects of adding acid in Example 5]

[0227] In an RO water suspension of Escherichia coli BLR(DE3) expressing PRT853 (HI: -0.68) in insoluble form, 1.8 μg / g wet cell deoxyribonuclease (SIGMA-ALDRICH) and 164 μg / g wet cell lysozyme (ThermoFisherScientific) were added, and the suspension was treated four times at room temperature using a high-pressure homogenizer (GEA, Pandaplus) at 600 bar to obtain a lysed suspension of the cells.

[0228] Acetic acid, citric acid, or sulfuric acid were added to the broken suspension at a concentration of 10–100 mM (see Table 8 for the relationship between sample number and acid concentration). The suspension was then centrifuged at 2500 × g for 30 seconds to confirm the aggregation of insoluble particles. Figure 6 These are photographs of the samples after centrifugation. Without the addition of acid (sample 1), it is impossible to obtain an insoluble aggregate by sedimentation under these low-speed centrifugation conditions. However, by adding acid, using any of the acid concentrations shown in Table 8, it is possible to obtain an aggregate of insoluble aggregates under these low-speed centrifugation conditions (samples 2-10).

[0229] The effect of acid addition on the insoluble particles after resuspending in RO water (centrifugation sedimentation resuspension) was also investigated. Specifically, the ruptured bacterial suspension was centrifuged (TOMYMX-305) at 11000×g for 5 minutes to obtain insoluble particles. These insoluble particles were relatively small particles that would require considerable centrifugation force and time to obtain without acid addition. After suspending these insoluble particles in RO water (centrifugation sedimentation resuspension), the acid concentrations shown in Table 8 were added as described above, and the mixture was centrifuged at 2500×g for 30 seconds to confirm the aggregation status of the insoluble particles. Figure 7 These are photographs of the samples after centrifugation. It can be confirmed that acid can effectively coagulate the centrifuged precipitate in the resuspension obtained by resuspending it in RO water. Furthermore, by resuspending in RO water, the particles are washed, and components from the culture medium and bacterial cells are removed. Therefore, compared to insoluble particles that are not resuspended in RO water, a cleaner-looking insoluble sample can be obtained.

[0230] [Table 8]

[0231] [Example 6: Effect of Acid Addition on Different Proteins in HI]

[0232] For the four insoluble substances with different hydrophobicities, namely PRT410 (HI: -0.81), PRT647 (HI: 0.04), PRT699 (HI: 0.17), and PRT698 (HI: 0.43), 5-30 mM citric acid was added using the same method as in Example 5, and the aggregation effect of the insoluble substances was confirmed as follows.

[0233] Deoxyribonuclease (1.8 μg / g wet cells) and lysozyme (164 μg / g wet cells) were added to an RO-water suspension of *E. coli* BLR(DE3) expressing various insoluble forms. The cells were homogenized four times at room temperature using an autoclave at 600 bar to disrupt the cell structure. After disruption, citric acid was added to the disrupted suspension at a concentration of 5–30 mM, and the suspension was centrifuged at 2500 × g for 30 seconds to confirm the aggregation status. Figure 8 These are photos of the samples after centrifugation.

[0234] Figure 8 In the table, 0, 5, 10, 20, and 30 represent the concentration (mM) of added citric acid (0 mM means no citric acid was added). H is the sample obtained by centrifuging the suspension without added citric acid at 11000 × g for 5 minutes.

[0235] Without acid, the insoluble form of any protein can only be obtained by centrifugation at 11000×g for 5 minutes. However, by adding citric acid of 10 mM or higher, the insoluble form of any protein can be obtained by low-speed centrifugation. Agglutination was also observed when 5 mM citric acid was added, but sedimentation was incomplete within a short time of 30 seconds (see reference). Figure 8 ).

[0236] The effect of acid addition was confirmed in proteins with different degrees of hydrophobicity, therefore it can be considered that this acid addition method can be applied to the aggregation of insoluble bodies of various proteins.

[0237] In addition, the protein recovery rates of the four precipitates PRT410, PRT647, PRT699, and PRT698, which were recovered by adding 10 mM citric acid and centrifuging at low speed, were calculated in the same manner as in Example 4. The results are shown in Table 9.

[0238] [Table 9]

[0239] Table 9 shows that for any protein, the insoluble body can be recovered without loss by adding acid.

[0240] Next, the purity of the recovered PRT410 insolubles was analyzed using SDS-PAGE. Figure 9 This is a photograph showing the results (electrophoresis results) of analyzing various treatment solutions of PRT410 using SDS-PAGE. Figure 9 In the electrophoresis process, molecular weight marker proteins were applied to lane M; acid-free fragmented suspensions were applied to lane 1; and insoluble bodies containing 10 mM citric acid were added to lane 2, resulting in protein concentrations of 1.5 μg. Staining after electrophoresis was performed using two methods: Oriole™ fluorescent gel staining agent (Bio-Rad) capable of staining all proteins, and InVision™ His-tagged intragel staining agent (Thermo Fisher Scientific) reacting with the His-tagged region of PRT410. PRT410, with a theoretical molecular weight of 53.6 kDa, was detected as a band near the 60 kDa molecular weight marker.

[0241] The electrophoretic bands of Oriole-stained gels were analyzed using a GelDoc EZ gel imaging system (manufactured by BIORAD) to calculate the purification purity of PRT410 in each treatment solution. The results are shown in Table 10.

[0242] [Table 10]

[0243] The purity of the precipitate fraction obtained without the addition of acid was 11.6%, while the addition of acid caused it to aggregate and precipitate densely, thus increasing the purity to 23.7% (reference). Figure 9 ).

[0244] It can be confirmed that the method of adding this acid is not only highly effective in the aggregation of insoluble bodies, but also an effective means of removing impurities from host cells.

[0245] It can be confirmed that the method of adding this acid is an excellent method as follows: there is no limitation on the type of insoluble protein. In addition, regardless of whether the insoluble body exists in the form of dense insoluble particles, it is an extremely effective means of agglomerating insoluble bodies and removing impurities from host cells, and can recover insoluble bodies with a very high yield.

[0246] [Example 7: Lipopolysaccharide (LPS) Removal Effect]

[0247] In *Escherichia coli*, used as the host cell, there exists LPS, a cell wall-derived substance unique to Gram-negative bacteria, known as endotoxin. Excessive amounts of this endotoxin are known to cause fever, multiple organ failure, and tachycardia; therefore, its reduction is preferred. The LPS content in insoluble bodies agglomerated by the addition of the metal salt or acid of this invention was measured to confirm the LPS-reducing effect.

[0248] The insoluble body (1) without the addition of metal salts and acids was obtained by the following method.

[0249] Specifically, 1.8 μg / g wet cell deoxyribonuclease (SIGMA-ALDRICH) and 164 μg / g wet cell lysozyme (Thermo Fisher Scientific) were added to an RO water suspension of E. coli BLR(DE3) expressing PRT853. The cells were homogenized four times at room temperature using a high-pressure homogenizer (GEA, Pandaplus) at 600 bar. After homogenization, the cells were centrifuged at 11000×g for 20 minutes using a TOMYMX-305 centrifuge. The precipitate fraction was then resuspended in RO water and treated at 11000×g for 30 minutes. This washing operation was performed twice. The resulting precipitate fraction was then resuspended in RO water and treated at 11000×g for 60 minutes to obtain insoluble fraction (1). The insoluble fraction (1) was obtained at 20°C.

[0250] (2) The insoluble body that has been aggregated by the addition of metal salts was obtained by the following method.

[0251] That is, the same method as above is used until the bacteria are broken up by treating them four times with a high-pressure homogenizer. After breaking up, 10 mM calcium chloride is added to cause the insoluble particles to coagulate, and the mixture is treated at 2500×g for 10 minutes. The resulting precipitate fraction is then resuspended in RO water and treated at 2500×g for 10 minutes. This washing operation is performed twice to obtain the insoluble particles (2) generated by the addition of metal salts in the form of precipitate fractions.

[0252] The insoluble body (3) that has been aggregated by acid addition is obtained by the following method.

[0253] That is, instead of adding metal salt, 10 mM citric acid is added, and otherwise, the insoluble body (3) produced by acid addition is obtained by the same method as when obtaining the insoluble body (2) with metal salt added.

[0254] The LPS content in these three insoluble substances was determined using the following method.

[0255] (i) Preparation of the test sample

[0256] Approximately 75 mg of each of the three insoluble samples (75.5 mg of insoluble (1), 75.1 mg of insoluble (2), and 75.0 mg of insoluble (3)) were weighed and added to a concentration of 50 mg / mL with distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) to prepare a suspension. After stirring with a vortex mixer, the pH was checked and adjusted to neutral by adding 5N sodium hydroxide aqueous solution (Wako Pure Chemical Industries, Ltd.). The insoluble sample was heated at 90°C for 20 minutes using a heater. After cooling, the sample was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected as the stock solution for analysis.

[0257] (ii) Determination of LPS content

[0258] Turbidimetric time analysis was performed using a Limulus ES-il Single Testwako (Wako Pure Chemical Industries, Ltd.) endotoxin analyzer (ET-6000 / J, Wako Pure Chemical Industries, Ltd.) according to the accompanying instructions. The CSE (E. coli UKT-B) included with the kit was used as the endotoxin standard. For each sample, the test stock solution was first diluted 1000 times before measurement. Insoluble particles (1) and (2) were measured after a 1000-fold dilution, but insoluble particle (3) was below the detection limit (<0.01 EU / mL), so the dilution ratio was changed to a 10-fold dilution to obtain the measured value. The results are shown in Table 11.

[0259] [Table 11]

[0260] In insolubles (2) and (3) obtained by adding metal salts or acids to cause them to agglomerate, the LPS content was found to be lower than that of insolubles (1) obtained without the addition of these substances. In particular, the LPS content was extremely low in insolubles (3) obtained by agglomeration using acid. The method of obtaining insolubles by adding metal salts or acids to cause them to agglomerate is also an excellent method for reducing LPS content.

[0261] [Example 8: Aggregation Effect Based on Anionic Flocculants]

[0262] In an RO aqueous suspension of *E. coli* BLR(DE3) expressing PRT853 (HI: -0.68) as insoluble particles, 1.8 μg / g wet cell deoxyribonuclease and 164 μg / g wet cell lysozyme were added. The cells were homogenized four times at room temperature using an autoclave to obtain a fragmented suspension. After homogenization, the flocculants shown in Table 12 were added to the fragmented suspension at a concentration of 0.05%. The sedimentation of the insoluble particles was confirmed by standing or centrifugation (2680 × g, 10 seconds). The results are shown in... Figure 10 .

[0263] Only anionic polyacrylate flocculants (Clifa PA-896) can effectively settle insoluble particles under either static or centrifugal separation conditions.

[0264] [Table 12]

[0265] [Example 9: Purity Improvement Effect Based on Anionic Flocculants]

[0266] The anionic flocculant Clifton PA-896, whose agglutination effect was confirmed in Example 8, was examined for its effect when added to the cell disruption suspension and the centrifuged sedimentation resuspension. The centrifuged sedimentation resuspension was obtained by centrifuging the disruption suspension at 11000 × g for 5 minutes at 20°C, and then resuspending the resulting precipitate fraction in RO water. The cell disruption suspension was obtained using the same method as in Example 8, except that it used Escherichia coli BLR(DE3) expressing PRT410 (HI: -0.81) in insoluble particulate form.

[0267] After adding the flocculant, the mixture was centrifuged at 2500×g for 5 minutes, and the purity of the insoluble particles in the resulting precipitate fraction was analyzed by SDS-PAGE. The results are shown below. Figure 11 . Figure 11In the process, a suspension of PRT410 cells freshly broken using a high-pressure homogenizer (broken suspension) is applied to lane 1; in lane 2, Cryoferrin PA-896 is added to the broken suspension at a concentration of 0.01% for precipitation, followed by centrifugation at 2500×g for 5 minutes to obtain the precipitate fraction; in lane 3, the broken suspension (lane 1) is centrifuged at 11000×g, 20°C, for 5 minutes to allow sedimentation, and the resulting precipitate fraction is resuspended in RO water to obtain a suspension (centrifuged sedimentation resuspension); in lane 4, Cryoferrin PA-896 is added to the centrifuged sedimentation resuspension at a concentration of 0.01%, followed by low-speed centrifugation at 2500×g for 5 minutes to obtain the precipitate fraction. Staining after electrophoresis used two methods: Oriole™ fluorescent gel staining agent (Bio-Rad) which stains all proteins, and InVision™ His-tagged intragel staining agent (Thermo Fisher Scientific) which reacts with the His-tagged region of PRT410. PRT410, with a theoretical molecular weight of 53.6 kDa, was detected as a band near the 60 kDa molecular weight marker.

[0268] Adding Cryptom PA-896 to the broken suspension at a rate of 0.01% caused the insoluble particles to aggregate, thereby enabling separation by low-speed centrifugation at 2500×g for 5 minutes. The purity of the resulting precipitate fraction increased from 12.2% to 53.5% (reference). Figure 11 Lanes 1 and 2, Table 13 (freshly broken). The suspension of freshly broken bacteria was centrifuged at 11000×g, 20℃, for 5 minutes using a high-pressure homogenizer. The resulting precipitate was then resuspended in RO water (centrifugation, sedimentation, and resuspension). This process increased the purity of the precipitate from 12.2% to 36.3% (reference). Figure 11 Lanes 1 and 3 (Table 13, Centrifuged Precipitate Resuspension) were used. Further, Cryptom PA-896 was added to the centrifuged precipitate resuspension at a rate of 0.01% to cause insoluble particles to aggregate, thereby enabling separation by low-speed centrifugation at 2500×g for 5 minutes. The purity of the resulting precipitate fraction was further increased from 36.3% to 51.8% (reference). Figure 11 Lanes 3 and 4, Table 13 (centrifugation sedimentation and resuspension)). Thus, the purity can be significantly improved by adding anionic flocculants.

[0269] [Table 13]

[0270] [Effects of Acid Addition, Heating, and Stirring in Example 10]

[0271] The insoluble form of PRT410 was used to confirm the agglutination effect induced by heating. Deoxyribonuclease 1.8 μg / g wet cells and lysozyme 164 μg / g wet cells were added to an RO-water suspension of *E. coli* BLR(DE3) expressing PRT410. The cells were homogenized four times at 600 bar at room temperature using an autoclave. After homogenization, the suspension was centrifuged at 2500 × g for 10 min, the supernatant was discarded, and the concentration was adjusted to 2.5 times. The concentrate was then diluted 2.5 times with RO-water. Citric acid was added to the homogenized suspension to reach 20 mM, and then heated and stirred as needed to obtain agglutinated samples. The samples were treated using the conditions described in Table 14. Heating was performed in a warm bath for the duration from the moment the bath reached 80°C. Stirring was carried out at 200 rpm. For the obtained aggregates, particle concentration and median particle size were determined using a CDA-1000 particle number analyzer (Sysmex Corporation). The results are shown in Table 14. Additionally, Figure 12 The frequency distribution and cumulative distribution of the median particle size are shown.

[0272] Sample X, which received neither acid nor stirring, Sample 1, which was heated without acid, and Sample 2, which was heated and stirred without acid, showed almost no agglomeration effect. On the other hand, Sample 3, which received only acid without heating / stirring, showed an increase in particle size compared to Samples X, 1, and 2. Furthermore, Sample 7, heated at 80°C for 2 hours, showed a further increase in particle size compared to Sample 3. For Samples 4-6, which were heated for different times and further stirred, the increase in particle size was further confirmed by stirring. While agglomeration depends on the processing time, the effect can be observed at least from 0.5 hours. This trend is due to… Figure 12 This has been proven. The sharper the peak of the particle size distribution, the higher the filterability; therefore, from... Figure 12 It was confirmed that adding acid and then heating and stirring improved the filterability.

[0273] [Table 14]

[0274] [Example 11: Improved filtration efficiency based on acid type]

[0275] The effect of acid addition on improving filtration was confirmed using the insoluble form of PRT410. Filtration was confirmed by adding acid; therefore, the filtration improvement effects of citric acid, hydrochloric acid, and sulfuric acid were compared based on the type of acid. The experimental methods were the same as in Example 10, except for the different acids. Acid treatment was performed at 80°C for 2 hours, with heating and stirring. Examples of the results regarding maximum filtration capacity and the relationship between filtration time and permeation flux are shown in Tables 15 and 16. The comparison results show that citric acid had the highest maximum filtration capacity and stable permeation flux, thus confirming its excellent industrial application.

[0276] [Table 15]

[0277] [Table 16]

[0278] [Example 12: Effect of Heating on Improved Protein Purity]

[0279] The purity of the insoluble forms of PRT799 (serial number 11, 200 kDa) and PRT587 (serial number 12, 100 kDa) after heating was analyzed by SDS-PAGE. Figure 13 and Figure 14 These are photographs showing the results (electrophoresis results) of the analysis of each treatment solution of PRT799 and PRT587 using SDS-PAGE. For each treatment solution, citric acid was added to adjust the pH to 3.75. Figure 13 In lane 1, a disrupted suspension heated to 80°C for 3 hours was applied; in lane 2, a non-heated disrupted suspension was applied. Regarding the disrupted suspension in lane 1, it was confirmed that heating decomposed contaminating proteins. Figure 14 Unheated fragmented suspension was applied to lane 1, while heated fragmented suspension was applied to lane 2 at 80°C for 2 hours. It was confirmed that the band near the 40 kDa molecular weight marker was broken down, and the detection intensity of the band (target protein) near the 100 kDa molecular weight marker in lane 2 was 1.2 times higher than that of the band in lane 1 without heating. Therefore, it was confirmed that heating improved the purity of the target protein.

[0280] [Effects of continuous heating in Example 13]

[0281] The insoluble form of PRT799 was used to confirm the agglutination effect induced by continuous heating. Deoxyribonuclease 1.8 μg / g wet cells and lysozyme 164 μg / g wet cells were added to an RO-water suspension of *E. coli* BLR(DE3) expressing PRT799. The cells were homogenized four times at room temperature using an autoclave at 600 bar. After homogenization, the suspension was centrifuged at 2500 × g for 10 minutes using a TOMYMX-305 centrifuge. The supernatant was discarded, and the solution was concentrated 2.5 times. The concentrate was then diluted 2.5 times with RO-water. Citric acid was added to the homogenized suspension at a concentration of 20 mM, followed by heating to obtain the agglutinated cells. All samples were processed using the conditions described in Table 17. Heating was performed using a MINIUHTT-20 continuous liquid sterilization system (Powerpoint International) at temperatures of 80°C, 85°C, 90°C, or 95°C, with heating times of 30 or 60 seconds for the broken suspension. The resulting aggregates were analyzed using a CDA-1000 particle size analyzer (Sysmex Corporation) to determine particle concentration and median particle size. The results are shown in Table 17. Additionally, Figure 14 and Figure 15 The frequency distribution and cumulative distribution of the median particle size are shown.

[0282] [Table 17]

[0283] According to the results in Table 17, when comparing the agglomerates of sample X obtained by heating in a warm bath at a temperature of 80°C for 2 hours with the agglomerates of samples 1, 2, 3, 4, 5, 6, 7, or 8 obtained by using a MINIUHTT-20 liquid continuous sterilization device (manufactured by Powerpoint International) at temperatures of 80°C, 85°C, 90°C, or 95°C for 30 seconds or 60 seconds, the particle size of the agglomerates obtained by using the MINIUHTT-20 liquid continuous sterilization device (manufactured by Powerpoint International) was at least equal to that of the agglomerates obtained by heating in a warm bath.

[0284] Depend on Figure 15 and Figure 16 It can also be confirmed that heating the broken suspension at high temperature for a short time can efficiently make the aggregate become huge.

[0285] [Example 14: Improved filtration effect through continuous heating]

[0286] The results for the filtration area and maximum filtration capacity of samples X, 5, 6, 7, and 8 are shown in Table 18. According to Table 18, the filtration performance when heated at high temperature for a short time using a liquid continuous sterilization device is equal to or better than that when heated in a warm bath for 2 hours, confirming that the filtration performance is improved by setting the heating of the broken suspension to high temperature and short time.

[0287] [Table 18]

Claims

1. A method for manufacturing a recombinant protein condensate, comprising the method of separating the insoluble form of the recombinant protein from recombinant cells expressing the recombinant protein intracellularly in the form of a condensate to manufacture the recombinant protein condensate, wherein, include: After the recombinant cells were ruptured, the insoluble forms of the recombinant proteins were agglutinated, and the resulting agglutinates were separated. Furthermore, the aggregation of the insoluble form of the recombinant protein is achieved by adding one or more selected from the group consisting of metal salts, acids, and anionic agglutinants.

2. The method for manufacturing the recombinant protein condensate as described in claim 1, wherein, The aggregation of the insoluble form of the recombinant protein is achieved by adding acid.

3. The method for manufacturing the recombinant protein condensate as described in claim 1, wherein, include: The recombinant protein condensate was separated by centrifugation at a force of less than 10,000 g.

4. The method for manufacturing the recombinant protein condensate as described in claim 1, wherein, include: The recombinant protein condensate is separated by natural sedimentation or filtration.

5. A method for manufacturing a recombinant protein condensate, comprising the following steps (A) to (C): (A) The step of lysing recombinant cells that express the target recombinant protein in the form of insoluble bodies to obtain a lysed suspension containing the recombinant protein in the form of insoluble bodies. (B) Add one or more of the group consisting of metal salts, acids and anionic coagulants to the broken suspension obtained in step (A) to cause the insoluble parts of the recombinant protein to aggregate, thereby obtaining a recombinant protein aggregate. (C) The step of separating the aggregates obtained in step (B) from the suspension.

6. The method for manufacturing the recombinant protein condensate as described in claim 5, wherein, In step (B), the aggregation of the insoluble form of the recombinant protein is carried out by adding acid.

7. The method for manufacturing the recombinant protein condensate as described in claim 5, wherein, Step (B) further includes heating.

8. The method for manufacturing the recombinant protein condensate as described in claim 7, wherein, Step (B) further includes stirring.

9. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The metal salt is selected from the group consisting of alkaline earth metal salts and earth metal salts.

10. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The acid is an oxyacid.

11. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The anionic coagulant is an anionic coagulant selected from the group consisting of polyacrylate, anionic polyacrylamide, and acrylamide-acrylate copolymer.

12. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The recombinant cells were broken down mechanically.

13. The method for manufacturing the recombinant protein condensate according to any one of claims 1 and 5 to 7, wherein, The recombinant protein condensate was separated by filtration.

14. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The recombinant cells are recombinant cells transformed from a host selected from the group consisting of bacteria, yeast, filamentous fungi, insect cells, plant cells, and animal cells.

15. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The recombinant protein is a structural protein.

16. The method for manufacturing the recombinant protein condensate according to any one of claims 1 to 7, wherein, The particle size of the recombinant protein condensate obtained by the inductive zone method was greater than 4 μm and less than 50 μm.

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